Method for detecting protein-protein interaction

ABSTRACT

A method for detecting an interaction between a first protein and a second protein comprises the steps of:
         expressing in a cell a first fusion protein comprising the first protein and an association-inducing protein, and a second fusion protein comprising the second protein and a fluorescent protein having a multimerization ability;   detecting a fluorescent focus formed by an association between the first fusion protein and the second fusion protein in the cell; and   determining an interaction between the first protein and the second protein according to the detection of the fluorescent focus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/JP2012/081539, filed on Dec. 5, 2012, which claims priority fromJapanese Patent Application No. 2011-266103, filed on Dec. 5, 2011, thecontents of all of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a method for detecting aprotein-protein interaction, applications thereof, and a kit for use inthe method.

BACKGROUND ART

Methods for detecting a protein-protein interaction can be roughlycategorized into two groups. One is a method characterized by using aprotein having been separated from living cells. Examples of such amethod include surface plasmon resonance, protein mass spectroscopy, andanisotropy measurements. However, these methods have difficultydetecting an interaction in an environment similar to an actualintracellular environment.

Then, as the second method, a method has been developed, in which aprotein-protein interaction is detected using living cells. Typicalmethods thereof are a yeast two hybrid system which detects atranscriptional activity of a reporter, and modified methods thereof.Besides, another method has been also developed, which utilizesreconstitution of enzymes such as β-galactosidase and dihydrofolatereductase (DHFR).

Nevertheless, these methods have problems that they are incapable ofdetecting a position where a protein-protein interaction has taken place(positional information on the protein-protein interaction), as well asa period until a protein-protein interaction takes place, a period untilthe interaction ends, a duration of the interaction, and the like(temporal information on the protein-protein interaction).

Meanwhile, the method for detecting a protein-protein interaction usingliving cells also includes a method utilizing reconstitution of afluorescent protein. Nevertheless, once reconstituted, the fluorescentprotein does not dissociate. Accordingly, this method has a problem thatit is incapable of detecting a period until a protein-proteininteraction ends, a duration of the interaction, and the like. Further,there is another problem that a period until a protein-proteininteraction takes place and the like cannot be detected because emissionof fluorescence requires a certain time after a protein-proteininteraction takes place.

Furthermore, there is also a method utilizing a luciferasereconstitution technique. In such a method, a luciferase is reversiblyreconstituted and dissociated. However, since the luminescent signalemitted by a reconstituted luciferase is weak, the exposure time has tobe a long in order to obtain intracellular positional information, andpositional information and temporal information on a protein-proteininteraction with high turnover rate cannot be obtained.

Additionally, in the methods utilizing the reconstitution of afluorescent protein, a luciferase, or the like, a signal can be detectedonly after such a reconstitution. This also brings about such a problemthat it is difficult to trace, for example, both before and after aprotein-protein interaction takes place, proteins which are located atdifferent positions by the interaction.

On the other hand, as a method for detecting a protein-proteininteraction in living cells, fluorescence resonance energy transfer(FRET) has been developed, which detects energy transfer dependent on adistance between molecules. This method has an advantage of obtainingpositional information and temporal information on where and when aprotein-protein interaction takes place. Nevertheless, since apositional relation between a donor fluorescent protein and an acceptorfluorescent protein used in the method is important to detect theprotein-protein interaction, the method involves a complicated step ofinvestigating the optimization of a linker (spacer) connecting thesefluorescent proteins to a detection-target protein, so that such asystem has been difficult to construct. Further, it has also beendifficult to analyze the result due to cross excitation by which anacceptor fluorescent protein is excited, and to bleed-through in whichfluorescence of a donor fluorescent protein bleeds through a filter(absorption filter) set for detecting fluorescence of an acceptorfluorescent protein. Moreover, use of fluorescent proteins of two colors(donor fluorescent protein and acceptor fluorescent protein) also bringsabout a problem that only limited fluorescent proteins are usable inorder to detect information other than a detection-target protein.

Recently, Tobias Meyer et al. have reported a method for detecting aprotein-protein interaction by utilizing intracellular localization(translocation) (PTL 1). In this method, one of proteins subjected tointeraction detection is fused to a protein that specifically binds to aparticular site in a cell, while the other of the proteins subjected tointeraction detection is fused to a fluorescent protein or the like.Then, these fusion proteins were expressed in a cell, and theprotein-protein interaction is detected on the basis of a signal of thefluorescent protein or the like at the particular site in the cell.

In addition, Nibert et al. have reported a method for detecting aprotein-protein interaction, using a fusion protein in which one ofproteins subjected to interaction detection is fused to a protein forforming a viral inclusion body, and using, as an indicator, accumulationof the other of the proteins subjected to interaction detection in theviral inclusion body (PTL 2).

However, in these methods for detecting a protein-protein interaction byutilizing intracellular localization, one of proteins subjected tointeraction detection is forcibly (artificially) translocated andconfined at a particular site in a cell. Accordingly, the detection isimpossible at a site where a protein-protein interaction naturally takesplace, that is, in an intracellular environment unique to theprotein-protein interaction, which brings about a problem thatpositional information on the protein-protein interaction cannot beobtained, and other similar problems. Moreover, it is also impossible todetect the interaction between proteins localized in a natural state atthe same site as the site of the intracellular localization.

Against this problem, Sara Peterson Bjorn et al. have reported a methodfor detecting a protein-protein interaction (redistribution-trapmethod), in which proteins are allowed to interact with each other in anintracellular environment where the proteins naturally function, andthen the cells are stimulated with a drug or the like to thereby induceaggregate formation from the interacting proteins, the aggregateformation being indicative of the interaction (PTL 3).

However, this method needs to stimulate cells at certain time so thatthe aggregate formation can be induced, and also needs to remove thedrug or the like used for the stimulation to detect the presence orabsence of an interaction subsequently after the stimulation. Hence, themethod has problems such as being incapable of obtaining temporalinformation on when the protein-protein interaction takes place, andincapable of detecting a protein-protein interaction that changes (takesplace, ends, takes place again, and so forth) for a certain period andat a certain position.

CITATION LIST Patent Literature

-   [PTL 1] International Publication No. WO2000/017221-   [PTL 2] International Publication No. WO2006/099486-   [PTL 3] U.S. Pat. No. 7,282,347

SUMMARY OF INVENTION Technical Problem

The present invention has been made in consideration of theabove-described problems in the conventional techniques. An object ofthe present invention is to provide a method capable of detecting aprotein-protein interaction in a cell in an intracellular environmentunique to the protein-protein interaction, and also capable of detectingpositional information and temporal information on the protein-proteininteraction.

Solution to Problem

The present inventors have come up with an idea of utilizing, indetecting an interaction between two proteins (a first protein and asecond protein), a first fusion protein comprising the first protein andan association-inducing protein, and a second fusion protein comprisingthe second protein and a fluorescent protein having a multimerizationability. Specifically, the inventors have come up with a construction ofa system as follows. When the two fusion proteins are expressed in acell, an interaction if any between the first protein and the secondprotein induces an association action between the association-inducingprotein and another association-inducing protein. Thereby, the fusionproteins autonomously form an assembly, and the fluorescent proteincontained in the fusion protein is detected as a fluorescent focus (seeFIGS. 1 and 2). Further, the inventors have come up with theutilization, as the association-inducing protein, of a protein havingsuch natures of: being present in a dispersed manner in a cell whenfused to monomeric Azami Green 1 (mAG1), one of monomeric fluorescentproteins; and forming a fluorescent focus (assembly) in a cell whenfused to a fluorescent protein having a multimerization ability.

Hence, the present inventors, first, expressed in cells candidateproteins fused to mAG1 or fluorescent proteins having a multimerizationability, and screened for association-inducing proteins on the basis offluorescent focus formation (see FIGS. 3 and 4).

The screening result has revealed that a PB1 domain of p62, a PB1 domainof TFG, a PB1 domain of PKCiota, a SAM domain of TEL, a SAM domain ofDGK delta, and a SAM domain of Tankyrase-1 are usable as theassociation-inducing protein.

Then, the present inventors have revealed that the use of theseidentified proteins as association-inducing proteins in combination withfluorescent proteins having a multimerization ability indeed enablesdetection of certain protein-protein interactions on the basis offluorescent foci.

This method is capable of detecting a protein-protein interaction in anintracellular environment unique to the protein interaction, and alsocapable of detecting positional information and temporal information onthe protein-protein interaction. Moreover, it is also possible toidentify an amino acid residue involved in the protein-proteininteraction, and to screen for a substance modulating theprotein-protein interaction.

Thus, the present invention relates to a method for detecting aprotein-protein interaction, applications thereof, and a kit for use inthe method. More specifically, the present invention provides thefollowing inventions.

(1) A method for detecting an interaction between a first protein and asecond protein, the method comprising the steps of:

expressing in a cell a first fusion protein comprising the first proteinand an association-inducing protein, and a second fusion proteincomprising the second protein and a fluorescent protein having amultimerization ability;

detecting a fluorescent focus formed by an association between the firstfusion protein and the second fusion protein in the cell; and

determining an interaction between the first protein and the secondprotein according to the detection of the fluorescent focus.

(2) The method according to (1), wherein the fluorescent focus isdetected to detect the interaction taking place or ending, a perioduntil the interaction takes place or ends, or a duration of theinteraction.

(3) The method according to (1), wherein the fluorescent focus isdetected to detect the interaction taking place or ending in response toa particular stimulus, a period until the interaction takes place orends, or a duration of the interaction.

(4) The method according to any one of (1) to (3) for screening for aprotein interacting with a particular protein, wherein

one of the first protein and the second protein is the particularprotein, while the other is a test protein, and

a protein interacting with the particular protein is selected accordingto the detection of the fluorescent focus.

(5) The method according to any one of (1) to (3) for identifying anyone of an amino acid residue in the first protein and an amino acidresidue in the second protein, which are involved in the interaction,wherein

in a case where a protein in which a mutation is introduced is used asany one of the first protein and the second protein, if a fluorescenceintensity of the fluorescent focus is reduced in comparison with aprotein in which no mutation is introduced, the amino acid residue inwhich the mutation is introduced is determined to be involved in theinteraction.

(6) A method for screening for a substance modulating an interactionbetween a first protein and a second protein, the method comprising thesteps of:

expressing in a cell a first fusion protein comprising the first proteinand an association-inducing protein, and a second fusion proteincomprising the second protein and a fluorescent protein having amultimerization ability, in presence of a test compound;

detecting a fluorescent focus formed by an association between the firstfusion protein and the second fusion protein in the cell; and

selecting the test compound as a substance inducing the interaction if afluorescence intensity of the fluorescent focus is higher than afluorescence intensity of a fluorescent focus formed in absence of thetest compound, and selecting the test compound as a substancesuppressing the interaction if the fluorescence intensity of thefluorescent focus is lower than the fluorescence intensity of thefluorescent focus formed in the absence of the test compound.

(7) The method according to any one of (1) to (6), wherein theassociation-inducing protein is at least one protein selected from thegroup consisting of a PB1 domain of p62, a PB1 domain of TFG, a PB1domain of PKCiota, a SAM domain of TEL, a SAM domain of DGK delta, and aSAM domain of Tankyrase-1.(8) A method for screening for an association-inducing protein, themethod comprising the steps of:

(a) expressing in a cell a fusion protein comprising a test protein andmAG1;

(b) expressing in a cell a fusion protein comprising the test proteinand a fluorescent protein having a multimerization ability; and

(c) selecting the test protein as an association-inducing protein if afluorescent focus is not detected in step (a) but a fluorescent focus isdetected in step (b).

(9) The method according to any one of (1) to (8), wherein thefluorescent protein having a multimerization ability is at least onefluorescent protein selected from the group consisting of monomericKusabira-Orange 2, Azami-Green, Kusabira-Orange 1, dimeric Keima-Red,Kikume Green-Red, monomeric Keima-Red, monomeric Midoriishi-Cyan1,monomeric Kusabira-Orange 1, monomeric Kikume Green-Red1,Midoriishi-Cyan1, Kusabira-Cyan1, dimeric Azami-Green (AB),dimericAzami-Green (AC), TGuv, Momiji, COR3.01, COR5, and DsRed2.(10) A kit for use in the method according to any one of (1) to (9), thekit comprising an instruction and at least one substance selected fromthe group consisting of the following (a) to (j):

(a) a vector comprising a DNA encoding the association-inducing proteinand a cloning site allowing an insertion of a DNA encoding a certainprotein in such a manner that the certain protein is fused to theassociation-inducing protein when expressed;

(b) a vector comprising a DNA encoding the fluorescent protein having amultimerization ability and a cloning site allowing an insertion of aDNA encoding a certain protein in such a manner that the certain proteinis fused to the fluorescent protein when expressed;

(c) a vector comprising a DNA encoding mAG1 and a cloning site allowingan insertion of a DNA encoding a certain protein in such a manner thatthe certain protein is fused to the mAG1 when expressed;

(d) a vector encoding the first fusion protein;

(e) a vector encoding the second fusion protein;

(f) a vector set comprising the vector according to any one of (a) and(d) and the vector according to any one of (b) and (e);

(g) a vector set comprising the vector according to (b) and the vectoraccording to (c);

(h) a transformed cell comprising a vector encoding the first fusionprotein;

(i) a transformed cell comprising a vector encoding the second fusionprotein; and

(j) a transformed cell comprising a vector encoding the first fusionprotein and a vector encoding the second fusion protein.

Advantageous Effects of Invention

The present invention makes it possible to detect a protein-proteininteraction in an intracellular environment unique thereto, and todetect positional information and temporal information on theprotein-protein interaction.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a diagram for illustrating a concept of a method for detectinga protein-protein interaction of the present invention. Specifically,the diagram illustrates that when a first fusion protein comprising afirst protein (B) and an association-inducing protein and a secondfusion protein comprising a second protein (A) and a fluorescent proteinhaving a multimerization ability are expressed in a cell, an interactionbetween the first protein (B) and the second protein (A) can bedetermined according to the detection of a fluorescent focus formed byassembly formation between the first fusion protein and the secondfusion protein in the cell.

FIG. 2 is a diagram for illustrating the concept of the method fordetecting a protein-protein interaction of the present invention.Specifically, the diagram illustrates that in a case where a firstprotein (C) does not interact with a second protein (A), even if a firstfusion protein comprising the first protein (C) and anassociation-inducing protein and a second fusion protein comprising thesecond protein (A) and a fluorescent protein having a multimerizationability are expressed in a cell, the first fusion protein and the secondfusion protein are not associated with each other, and are present in adispersed manner in the cell, so that no fluorescent focus is detected.

FIG. 3 is a diagram for illustrating a concept of a screening method foran association-inducing protein according to the present invention.Specifically, the diagram illustrates that an association-inducingprotein according to the present invention is capable of forming anassembly (fluorescent focus) in a cell, when fused to a fluorescentprotein having a multimerization ability.

FIG. 4 is a diagram for illustrating the concept of the screening methodfor an association-inducing protein according to the present invention.Specifically, the diagram illustrates that the association-inducingprotein according to the present invention is present in a dispersedmanner in a cell, when fused to monomeric Azami Green 1 (mAG1).

FIG. 5 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a protein (mAG1-p62(PB1)) composed of mAG1 fused to a PB1domain of p62 (p62(PB1)), and a protein (AG-p62(PB1)) composed ofp62(PB1) fused to Azami Green (AG) serving as the fluorescent proteinhaving a multimerization ability.

FIG. 6 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a fusion protein composed of a FRB domain of a mTORprotein and an AG protein, and a fusion protein composed of p62(PB1) andFKBP12. Note that a FRB domain of a mTOR protein (mTOR(FRB)) and aFKBP12 protein are known to interact with each other in the presence ofrapamycin.

FIG. 7 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a fusion protein composed of a FRB domain of a mTORprotein and p62(PB1), and a fusion protein composed of an AG protein andFKBP12. Note that, in the figure, the scale bars at the lower rightportions represent 5 μm.

FIG. 8 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells the fusion protein composed of a FRB domain of a mTORprotein and p62(PB1), and a fusion protein composed of a mAG1 proteinand FKBP12. Note that, in the figure, the scale bars at the lower rightportions represent 5 μm.

FIG. 9 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a fusion protein (mTOR-AG) composed of a FRB domain of amTOR protein and an AG protein, and a fusion protein (p62(PB1)-FKBP12)composed of a p62(PB1) protein and FKBP12, in the absence (−) orpresence (+) of rapamycin.

FIG. 10 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a fusion protein (mTOR-mAG1) composed of a FRB domain ofa mTOR protein and a mAG1 protein, and the fusion protein(p62(PB1)-FKBP12) composed of a p62(PB1) protein and FKBP12, in theabsence (−) or presence (+) of rapamycin.

FIG. 11 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells the fusion protein (mTOR-AG) composed of a FRB domain ofa mTOR protein and an AG protein, and a fusion protein(p62(PB1_nc)-FKBP12) composed of a p62(PB1) protein mutant having lostno homomultimerization ability and FKBP12, in the absence (−) orpresence (+) of rapamycin.

FIG. 12 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a protein composed of a mAG1 protein or an AG protein(fluorescent protein having a multimerization ability) fused to a PB1domain of MEK5 (MEK5(PB1)) or a PB1 domain of Nbr1 (Nbr1(PB1)).

FIG. 13 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a protein composed of a mAG1 protein or an AG proteinfused to a PB1 domain of PKCiota (PKCiota(PB1)) or a PB1 domain of TFG(TFG(PB1)).

FIG. 14 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a protein composed of a mAG1 protein or an AG proteinfused to a SAM domain of TEL (TEL (SAM)) or a SAM domain of DGK delta(DGKd) (DGK delta(SAM)).

FIG. 15 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a protein composed of a mAG1 protein or an AG proteinfused to a SAM domain of Tankyrase (Tankyrase(SAM)) or a SAM domain ofEphB2 (EphB2(SAM)).

FIG. 16 shows micrographs for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells the following fusion-protein combinations, in thepresence of rapamycin: a combination of a fusion protein (mTOR(FRBdomain)-AG) composed of a FRB domain of a mTOR protein and an AG proteinwith a fusion protein (FKBP12-TFG(PB1)) composed of a FKBP12 protein andTFG(PB1); a combination of mTOR(FRB domain)-AG with a fusion protein(FKBP12-TEL(SAM)) composed of a FKBP12 protein and TEL(SAM); acombination of mTOR(FRB domain)-AG with a fusion protein(FKBP12-DGKd(SAM)) composed of a FKBP12 protein and DGK delta (SAM); anda combination of mTOR(FRB domain)-AG with a fusion protein(FKBP12-Tankyrase(SAM)) composed of a FKBP12 protein and Tankyrase(SAM).

FIG. 17 shows micrographs for illustrating the result of analyzingwhether or not it is possible to detect a rapamycin-dependentinteraction between mTOR(FRB) and a FKBP12 protein according tofluorescent focus formation by using KO1, dKeima, KikGR, or AG as thefluorescent protein having a multimerization ability according to thepresent invention.

FIG. 18 shows a micrograph for illustrating the result of analyzing thepresence or absence of fluorescent focus formation by expressing incultured cells a protein composed of monomeric Kusabira-Orange 2 (mKO2)fused to p62(PB1).

FIG. 19 shows micrographs for illustrating that the result of analyzingwhether or not a fluorescence intensity of a fluorescent focus formed byan association between mTOR(FRB domain)-AG and FKBP12-p62(PB1) isdependent on the rapamycin concentration.

FIG. 20 is a graph for illustrating that the result of analyzing whetheror not the fluorescence intensity of the fluorescent focus formed by theassociation between mTOR(FRB domain)-AG and FKBP12-p62(PB1) is dependenton the rapamycin concentration.

FIG. 21 is a graph for illustrating that the result of analyzing whetheror not a fluorescence intensity of a fluorescent focus formed by anassociation between mTOR(FRB domain)-AG and p62(PB1)-FKBP12 is dependenton the rapamycin concentration.

FIG. 22 is a graph for illustrating the result of analyzing whether ornot the fluorescence intensity of the fluorescent focus formed by theassociation between mTOR(FRB domain)-AG and p62(PB1)-FKBP12 in thepresence of rapamycin is suppressed in a manner dependent on theconcentration FK506. Note that FK506 competitively inhibits between aninteraction between a FKBP12 protein and rapamycin, thereby inhibitingan interaction between the FKBP12 protein and a FRB domain of a mTORprotein (mTOR(FRB)).

FIG. 23 shows micrographs for illustrating the result of analyzing afluorescence intensity of a fluorescent focus in the presence ofNutlin-3 by expressing in cultured cells a fusion protein (p62(PB1)-p53)composed of p62(PB1) and a portion of p53, and a fusion protein(AG-MDM2) composed of an AG protein and MDM2. Note that Nutlin-3 isknown as an inhibitor against an interaction between a p53 protein andan MDM2 protein. Moreover, in the figure, the scale bars at the lowerleft portions represent 10 μm.

FIG. 24 is a graph for illustrating the result of analyzing whether ornot the fluorescence intensity of the fluorescent focus formed by theassociation between p62(PB1)-p53 and AG-MDM2 is suppressed in a mannerdependent on the Nutlin-3 concentration.

FIG. 25 is a graph for illustrating the result of analyzing whether ornot the fluorescence intensity of the fluorescent focus formed by theassociation between p62(PB1)-p53 and AG-MDM2 is suppressed in a mannerdependent on the Nutlin-3 concentration.

FIG. 26 shows micrographs for illustrating the result of observing cellsexpressing p62(PB1)-p50 and AG-p65 (IκBα (−)), and cells expressingp62(PB1)-p50, AG-p65, and IκBα (IκBα (+)). Note that p50 and p65 form aheterodimer, which is localized in the nucleus, and also localized inthe cytoplasm by the interaction with IκBα. Moreover, in the figure,“AG” shows the result of detecting AG-derived fluorescence.“Immunostaining with anti-IκBα antibody” shows the result of observingcells subjected to this immunostaining. “Merging” shows the result ofmerging the “AG”, the “immunostaining with anti-IκBα antibody,” and theresult of observing cells whose nuclei were stained with Hoechst 33342.

FIG. 27 is a graph for illustrating the result of analyzing whether ornot intracellular localization of a fluorescent focus formed by anassociation between p62(PB1)-p50 and AG-p65 is changed in accordancewith the amount of IκBα introduced to the cells (the amount of pIκBαadded).

FIG. 28 shows micrographs for illustrating the result of observing cellsco-expressing p62(PB1)-CDK4 and AG-p21 (AG-p21+PB1-CDK4), and cellsexpressing p62(PB1)-CDK4, AG-p21, and Cyclin D1 (AG-p21+PB1-CDK4+CyclinD1). Note that p21 interacts with a complex composed of CDK4 and CyclinD1. Moreover, in the figure, “AG” shows the result of detectingAG-derived fluorescence. “Immunostaining with anti-Cyclin D1 antibody”shows the result of observing cells subjected to this immunostaining.“Merging” shows the result of merging the “AG” and the “immunostainingwith anti-Cyclin D1 antibody.”

FIG. 29 shows micrographs for illustrating the result of analyzing afluorescence intensity and localization of a fluorescent focus byexpressing in cultured cells the following fusion-protein combinations.Note that, in the figure, the scale bars at the lower right portionsrepresent 20 μm: a combination of a fusion protein (p62(PB1)-Sec5)composed of p62(PB1) and a portion of a Sec5 protein with a fusionprotein (AG-RalB(WT)) composed of an AG protein and a RalB protein(wildtype); a combination of p62(PB1)-Sec5 with a fusion protein(AG-RalB(S28N)) composed of an AG protein and a RalB protein (inactivemutant); and a combination of p62(PB1)-Sec5 with a fusion protein(AG-RalB(Q72L)) composed of an AG protein and a RalB protein (activemutant). Note that a Sec5 protein is known to interact with a RalBprotein in a GTP-activated form. Moreover, it is known that theinteraction is reduced with an inactive mutant RalB(S28N) of RalB, butenhanced with an active mutant RalB(Q72L) of RalB. Further, it has alsobeen revealed that a RalB protein is localized at the cell membrane bypalmitoylation of the C-terminus thereof.

FIG. 30 shows micrographs for illustrating the result of detectingfluorescence only in the vicinity of the cell membrane by expressing incultured cells the following fusion-protein combinations: a combinationof p62(PB1)-Sec5 with AG-RalB(WT); a combination of p62(PB1)-Sec5 withAG-RalB(Q72L); a combination of p62(PB1)-Sec5 with AG-RalB(S28N); and acombination of p62(PB1) with AG-RalB(WT).

FIG. 31 shows micrographs for illustrating the result of observing cells(WT) co-expressing p62(PB1)-p53 and AG-MDM2, and cells (W23L)co-expressing p62(PB1)-p53_W23L and AG-MDM2. Note that the amino acid atposition 23 of p53 is located at an interaction interface site betweenp53 and MDM2, and a W23L mutation of p53 reduces the interaction.

FIG. 32 shows micrographs for illustrating the result of observingformation and extinction of a fluorescent focus formed by an associationbetween a fusion protein (Calmodulin-AG) composed of a calmodulinprotein and an AG protein, and a fusion protein composed(M13peptide-p62(PB1)) of a partial sequence (M13 peptide) of myosinlight chain kinase 2 and p62(PB1), before histamine addition, 90 secondsafter the addition, and 620 seconds after the addition. Note that, inthe figure, the scale bars at the lower right portions represent 5 μm.Moreover, it has been revealed that an interaction between calmodulinand an M13 peptide takes place in response to a transient increase inintracellular calcium ion concentration that occurs when a Gprotein-coupled receptor (GPCR) receives a ligand (for example,histamine).

FIG. 33 shows micrographs for illustrating the result of observinglocalization of a fluorescent focus formed by an association betweenFKBP12-p62(PB1) and a fusion protein (mTOR(FRB domain)-AGNLS) composedof mTOR(FRB domain), an AG protein, and a nuclear localization signal(NLS). Note that, from the left, the first panel shows a fluorescenceimage of cells before rapamycin addition, the second panel shows afluorescence image of the cells after the rapamycin addition, and thethird panel shows the result of merging superimposing the fluorescenceimage and bright-field image of the cells after the rapamycin addition.Moreover, in the figure, the scale bars at the lower right portionsrepresent 5 μm.

FIG. 34 shows a micrograph for illustrating the result of observinglocalization of a fluorescent focus formed by an association between afusion protein (AG-cRaf) composed of an AG protein and a cRaf protein,and a fusion protein (p62(PB1)-HRas) composed of p62(PB1) and an HRasprotein, the p62(PB1)-HRas having a prenylated sequence at theC-terminus. Note that, in the figure, the scale bar at the lower rightportion represents 5 μm.

FIG. 35 shows a micrograph for illustrating the result of observinglocalization of a fluorescent focus formed by an association between afusion protein (Smac-p62(PB1)) composed of a portion of a Smac proteinand p62(PB1), and a fusion protein (XIAP-AG) composed of a portion of aXIAP protein and an AG protein. Note that, in the figure, the scale barat the lower right portion represents 5 μm.

FIG. 36 shows a micrograph for illustrating the result of observinglocalization of a fluorescent focus formed by an association between afusion protein (p62(PB1)-BclX(L)) composed of p62(PB1) and a portion ofa BclX(L) protein, and a fusion protein (AG-BAD) composed of an AGprotein and a portion of a BAD protein. Note that, in the figure, thescale bar at the lower right portion represents 5 μm.

FIG. 37 shows a micrograph for illustrating that a fluorescent focusformed by an association between a fusion protein (AG-Rac1) composed ofan AG protein and a Rac1 protein, and a fusion protein (p62(PB1)-PBD)composed of p62(PB1) and a p21 binding domain is localized in thenucleus. Note that, in the figure, the scale bar at the lower rightportion represents 5 μm. Moreover, guanine nucleotide exchange factors(GEFs) convert a Rac1 protein to an active form. Further, it is knownthat an active Rac1 protein and PBD interact with each other.Furthermore, GEFs are localized differently depending on the type. Forthis reason, an interaction between a Rac1 protein and PBD takes placein intracellular regions in accordance with the GEFs localizeddifferently depending on the type.

FIG. 38 shows micrographs for illustrating that a fluorescent focusformed by an association between AG-Rac1 and p62(PB1)-PBD is localizedat the border of cells. Note that, in the figure, the lower panel isobtained by enlarging a region surrounded by the white line in the upperpanel. Moreover, the scale bar at the lower right portion of the upperpanel represent 5 μm, and the scale bar at the upper left portion of thelower panel represents 1 μm.

FIG. 39 shows micrographs for illustrating that when AG-Rac1 andp62(PB1) are expressed in cells, these proteins do not interact witheach other, so that no fluorescent focus is detected at the border andso on of a cell. Note that, in the figure, the lower panel is obtainedby enlarging a region surrounded by the white line in the upper panel.Moreover, the scale bar at the lower right portion of the upper panelrepresents 5 μm, and the scale bar at the upper left portion of thelower panel represents 1 μm.

FIG. 40 shows micrographs for illustrating the result of observing cellsexpressing AG-Rac1 and p62(PB1)-PBD in the absence (−) or presence (+)of an inhibitor mevastatin against geranylgeranyl group modification.Note that if geranylgeranyl group modification is inhibited, Rac1 islocalized in the nucleus. Moreover, the figure shows the results ofobserving the same cells; “A” is observed using a normal invertedepifluorescence microscope; and “B” is observed using a total internalreflection fluorescence microscopy system with arc lamp source capableof exciting only the vicinity of the cell membrane.

FIG. 41 shows micrographs for illustrating the result of observing cellsexpressing AG-Rac1 and RhoGDI-p62(PB1) in the absence (−) or presence(+) of an inhibitor mevastatin against geranylgeranyl groupmodification. Note that a Rac1 protein interacts with RhoGDI via ageranylgeranyl group of the Rac1 protein. Moreover, the figure shows theresults of observing the same cells; “A” is observed using a normalinverted epifluorescence microscope; and “B” is observed using a totalinternal reflection fluorescence microscopy system with arc lamp sourcecapable of exciting only the vicinity of the cell membrane.

FIG. 42 shows micrographs for illustrating the result of detecting onlyfluorescence in the vicinity of the cell membranes of cells (WT)expressing p62(PB1)-KRas(WT) and AG-cRaf(R59A), and cells (G12D)expressing p62(PB1)-KRas(G12D) and AG-cRaf(R59A), after EGF addition (+)or no addition (−). Note that KRas activated in a manner dependent onEGF interacts with cRaf. Moreover, this protein-protein interactionchanges the localization of cRaf from the cytoplasm to the cellmembrane.

FIG. 43 is a graph for illustrating a change over time in a totalfluorescence intensity of fluorescent foci in cells co-expressingp62(PB1)-BclX(L) and Bak-AG after ABT-737 addition. Note that althoughBclX(L) and Bak interact with each other via a BH3 domain, thisprotein-protein interaction is competitively inhibited by ABT-737 (BH3mimetic).

FIG. 44 is a graph for illustrating a change over time in a totalfluorescence intensity of fluorescent foci in cells co-expressingp62(PB1)-BclX(L) and AG-Bax, after ABT-737 addition. Note that althoughBclX(L) and Bax interact with each other via a BH3 domain, thisprotein-protein interaction is competitively inhibited by ABT-737.

FIG. 45 is a graph for illustrating a change over time in a totalfluorescence intensity of fluorescent foci in cells stably expressingp62(PB1)-p53 and AG-MDM2, before and after Nutlin-3 addition. Note that,in the graph, the x axis represents time (minutes), provided that timewhen Nutlin-3 was added is 0.

FIG. 46 is a graph for illustrating a change over time in a totalfluorescence intensity of fluorescent foci in cells stably expressingmTOR(FRB domain)-AG and p62(PB1)-FKBP12, before and after rapamycinaddition. Note that, in the graph, the x axis represents time (minutes),provided that time when rapamycin was added is 0.

FIG. 47 is a graph for illustrating a change over time in a totalfluorescence intensity of fluorescent foci in cells expressingp62(PB1)-ERK_substrate and AG-Pin1(ww)-NES, before and after EGF andU0126 addition. Note that when ERK in cells is activated by an EGFstimulus, the ERK substrate (ERK_substrate) is phosphorylated; as aresult, the ERK_substrate and a ww domain of a Pin1 protein (Pin1(ww))interact with each other. Further, if a MEK inhibitor U0126 is added,the ERK activity is decreased; as a result, the ERK substrate isdephosphorylated, terminating the interaction between the ERK substrateand Pin1(ww). In the graph, the x axis represents time (minutes),provided that time when EGF was added is 0. Additionally, U0126 wasadded to the cells, 14 minutes after the EGF addition.

FIG. 48 is a graph for illustrating a change over time in a totalfluorescence intensity of fluorescent foci in cells expressingp62(PB1)-HRas(WT) and AG-cRaf(R59A), before and after EGF addition. Notethat, in the graph, the x axis represents time (minutes), provided thattime when EGF was added is 0.

FIG. 49 shows micrographs for illustrating the result of observing cellsexpressing AG-mCAB, p62(PB1)-FKBP12, and mTOR(FRB)-KO1, in the absence(−) or presence (+) of rapamycin, or in the absence (−) or presence (+)of FK506. Note that rapamycin binds to a FKBP12 protein, forming acomplex, and further that this complex binds to a FRB domain of a mTORprotein (mTOR(FRB)). Moreover, mCAB (protein composed of a portion ofcalcineurin A fused to a portion of calcineurin B) interacts with aFKBP12 protein via FK506. In the figure, “AG” and “KO1” show the resultsof detecting fluorescences derived from AG and KO1, respectively.

DESCRIPTION OF EMBODIMENTS Method for Detecting Protein-ProteinInteraction

A method for detecting a protein-protein interaction of the presentinvention is a method for detecting an interaction between a firstprotein and a second protein, the method comprising the steps of:

expressing in a cell a first fusion protein comprising the first proteinand an association-inducing protein, and a second fusion proteincomprising the second protein and a fluorescent protein having amultimerization ability;

detecting a fluorescent focus formed by an association between the firstfusion protein and the second fusion protein in the cell; and

determining an interaction between the first protein and the secondprotein according to the detection of the fluorescent focus.

In the present invention, the term “protein” means a molecule in which 2or more amino acids are linked by a peptide bond(s), and modifiedproducts thereof. Thus, the term is a concept including not onlyfull-length proteins, but also so-called oligopeptides and polypeptides.Examples of the modification of the protein include phosphorylation,glycosylation, palmitoylation, prenylation (for example,geranylgeranylation), methylation, acetylation, ubiquitination,SUMOylation, hydroxylation, and amidation.

As the “first protein” and the “second protein” according to the presentinvention, it is possible to use desired proteins intended for detectionof interaction.

The “interaction between the first protein and the second protein”according to the present invention includes not only directinteractions, but also indirect interactions such as an interaction forforming a complex in which another molecule (protein, nucleic acid,sugar, lipid, low-molecular-weight compound, or the like) is interposedbetween the first protein and the second protein.

The “fluorescent protein having a multimerization ability” according tothe present invention is a fluorescent protein capable of forming afluorescent focus, when fused to a PB1 domain of p62 (p62(PB1)) andexpressed in a cell, as a result that the fusion proteins with thep62(PB1) are associated with each other. Thus, the “fluorescent proteinhaving a multimerization ability” includes not only fluorescent proteinscapable of forming a homomultimer in a cell without fusing withp62(PB1), but also fluorescent proteins such as mKO2 generally believedto be monomeric fluorescent proteins, as described in Examples later.Examples of such a “fluorescent protein having a multimerizationability” include Midoriishi-Cyan1 (MiCy1), Kusabira-Orange 1 (KO1),dKeima570 (dimeric Keima570), dimeric Keima-Red (dKeima, dKeima-Red),Azami-Green (AG), Kaede, Kikume Green-Red (KikGR, KikGR1), monomericKusabira-Orange 1 (mKO1), monomeric Kusabira-Orange 2 (mKO2), TurboGFP,TurboYFP, ZsGreen, DsRed, HcRed, eqFP578, eqFP611, EosFP, FP484, RenillaGFP, Dendra, IFP1.4, iRFP, monomeric Keima-Red (mKeima, mKeima-Red),monomeric Midoriishi-Cyan1 (mMiCy1), monomeric Kikume Green-Red1(mKikGR1), Kusabira-Cyan1 (KCy1), dimeric Azami-Green (AB) (dAG (AB)),dimeric Azami-Green (AC) (dAG (AC)), TGuv, Momiji, COR3.01, COR5, andDsRed2. Preferable are mKO2, mKeima, mMiCy1, mKO1, mKikGR1, MiCy1, KCy1,KO1, dKeima, dAG (AB), dAG (AC), TGuv, Momiji, KikGR, AG, COR3.01, COR5,and DsRed2. Moreover, from the viewpoint of facilitating detection of aclear fluorescent focus in the method of the present invention, morepreferable are fluorescent proteins capable of forming a homotetramer,such as TGuv, Momiji, AG, KikGR, COR3.01, COR5, and DsRed2, andparticularly preferable is AG.

Note that, typically, mKO2, AG, KO1, dKeima, KikGR, mKeima, mMiCy1,mKO1, mKikGR1, MiCy1, KCy1, dAG (AB), dAG (AC), TGuv, Momiji, COR3.01,DsRed2, and COR5 are respectively a protein having the amino acidsequence of SEQ ID NO: 133, a protein having the amino acid sequencespecified under Genbank ACCESSION No: AB107915, a protein having theamino acid sequence specified under Genbank ACCESSION No: AB128820, aprotein having the amino acid sequence specified under Genbank ACCESSIONNo: AB209968, a protein having the amino acid sequence specified underGenbank ACCESSION No: AB193293, a protein having the amino acid sequencespecified under Genbank ACCESSION No: AB209969, a protein having theamino acid sequence of SEQ ID NO: 137, a protein having the amino acidsequence specified under Genbank ACCESSION No: AB128821, a proteinhaving the amino acid sequence of SEQ ID NO: 139, a protein having theamino acid sequence specified under Genbank ACCESSION No: AB128822, aprotein having the amino acid sequence of SEQ ID NO: 141, a proteinhaving the amino acid sequence of SEQ ID NO: 143, a protein having theamino acid sequence of SEQ ID NO: 145, a protein having the amino acidsequence of SEQ ID NO: 147, a protein having the amino acid sequence ofSEQ ID NO: 149, a protein having the amino acid sequence of SEQ ID NO:151, a protein having the amino acid sequence of SEQ ID NO: 153, and aprotein having the amino acid sequence of SEQ ID NO: 172.

The amino acid sequences of these fluorescent proteins may be mutatednaturally (i.e., non-artificially). Moreover, a mutation can also beintroduced artificially. Such a mutant can also be used in the presentinvention, as long as it can emit fluorescence and form a homomultimerin a cell.

The “association-inducing protein” according to the present invention isa protein allowing a fluorescent focus to be detected when fused to afluorescent protein having a multimerization ability and expressed in acell, as a result that such fusion proteins are associated with eachother, and being present in a dispersed state in a cell when fused tomonomeric Azami Green 1 (mAG1) and expressed, as illustrated in Examplesand FIGS. 3 and 4 later.

The “association-inducing protein” according to the present invention ispreferably a PB1 domain of p62, a PB1 domain of TFG, a PB1 domain ofPKCiota, a SAM domain of TEL, a SAM domain of DGK delta, and a SAMdomain of Tankyrase-1. From the viewpoint of facilitating detection of afluorescent focus in the method of the present invention, morepreferable are a PB1 domain of p62, a PB1 domain of TFG, a SAM domain ofTEL, a SAM domain of DGK delta, and a SAM domain of Tankyrase-1.

In addition, typically, a PB1 domain of p62, a PB1 domain of TFG, a PB1domain of PKCiota, a SAM domain of TEL, a SAM domain of DGK delta, and aSAM domain of Tankyrase-1 are respectively a protein having the aminoacid sequence specified under SEQ ID NO: 4, a protein having the aminoacid sequence specified under SEQ ID NO: 12, a protein having the aminoacid sequence specified under SEQ ID NO: 10, a protein having the aminoacid sequence specified under SEQ ID NO: 14, a protein having the aminoacid sequence specified under SEQ ID NO: 18, and a protein having theamino acid sequence specified under SEQ ID NO: 20.

The amino acid sequences of these “association-inducing proteins” may bemutated naturally (i.e., non-artificially). Moreover, a mutation canalso be introduced artificially. Such a mutant having no associationability by itself can also be used in the present invention, as long asit has such a nature of forming an assembly (fluorescent focus) whenfused to a fluorescent protein having a multimerization ability.

In the “first fusion protein” according to the present invention, theassociation-inducing protein may be fused on either the N-terminus sideor the C-terminus side of the first protein. Further, theassociation-inducing protein may be fused to the first protein directly,or may be fused indirectly via a spacer protein. Furthermore, the “firstfusion protein” according to the present invention may be fused toanother functional protein. In this case, the other functional proteinmay be fused on one or both of the N-terminus side and the C-terminusside of the fusion protein, or may be fused directly or indirectlybetween the association-inducing protein and the first protein. Theother functional protein is not particularly limited, and selected asappropriate depending on a function desirably provided to the fusionprotein according to the present invention. Examples of a functionalprotein used to facilitate purification of the fusion protein include aMyc-tag protein, a His-tag protein, a hemagglutin (HA)-tag protein, aFLAG-tag protein (registered trademark, Sigma-Aldrich Co.), aglutathione S-transferase (GST) protein, and also a fluorescent proteinthat exhibits wavelength characteristics different from those of thefluorescent protein having a multimerization ability in the secondfusion protein.

In the “second fusion protein” according to the present invention, thefluorescent protein having a multimerization ability may be fused oneither the N-terminus side or the C-terminus side of the second protein,as in the case of the “first fusion protein.” Moreover, the fluorescentprotein having a multimerization ability may be fused to the secondprotein directly, or may be fused indirectly via the spacer protein.Furthermore, the “second fusion protein” according to the presentinvention may be fused to the above-described other functional protein.In this case, the other functional protein may be fused on one or bothof the N-terminus side and the C-terminus side of the fusion protein, ormay be fused directly or indirectly between the fluorescent proteinhaving a multimerization ability and the second protein, as in the caseof the “first fusion protein.”

The “cell” according to the present invention are not particularlylimited, and may be an eukaryotic cell, or may be a prokaryotic cell.Examples of the “cell” include an animal cell (HeLaS3 cell, U2OS cell,and the like), an insect cell (Sf9 cell, and the like), a plant cell,yeast, and Escherichia coli. Moreover, such cells may be in a state ofbeing cultured in vitro (for example, cells grown in a medium or on amedium), or in a state of being present in vivo (for example, cells in atransgenic animal in which a DNA encoding the first fusion protein and aDNA encoding the second fusion protein are introduced).

The expression of the fusion proteins in the cell according to thepresent invention may be a transient expression or a constitutiveexpression, depending on the purpose. The fusion proteins in the cellcan be expressed by introducing into the cell a vector according to thepresent invention, which will be described later. Examples of knowntechniques for introducing the vector into the cell include, in the caseof an animal cell, a lipofection method, an electroporation method, aphosphate calcium method, a DEAE-dextran method, and methods utilizing avirus (adenovirus, lentivirus, adeno-associated virus, or the like).Moreover, in the case of an insect cell, the examples include methodsutilizing a baculovirus. Further, in the case of a plant cell, theexamples include an Agrobacterium method, an electroporation method, alithium acetate method, and the like. In addition, in the case ofyeasts, the examples include a lithium acetate method, anelectroporation method, and a spheroplast method. Furthermore, in thecase of Escherichia coli, the examples include a heat shock method (forexample, a calcium chloride method, a rubidium chloride method), anelectroporation method, and the like.

A “fluorescent focus” to be detected in the present invention is formedby an association between the first fusion protein and the secondprotein. Typically, the “fluorescent focus” has a fluorescence intensityin a region of 0.2 to 5 μm, the fluorescence intensity being higher thana fluorescence intensity of a fluorescent protein having amultimerization ability, which is present in a dispersed state (seeExamples described later and FIGS. 1 and 2).

The “detection of the fluorescent focus” can be carried out, forexample, through observation using a fluorescence microscope includingan excitation filter and an absorption filter corresponding to afluorescent protein having a multimerization ability, and analysis usingan imaging cytometer such as IN Cell Analyzer (manufactured by GEHealthcare).

In the method of the present invention, if the fluorescent focus isdetected in the cell, it can be determined that the first protein andthe second protein interact with each other; meanwhile, if thefluorescent focus is not detected, it can be determined that the firstprotein and the second protein do not interact with each other.

<Screening Method for Association-Inducing Protein>

The association-inducing protein according to the present invention canbe selected, as described in Examples later, by a screening methodcomprising the following steps (a) to (c):

(a) expressing in a cell a fusion protein comprising a test protein andmAG1;

(b) expressing in a cell a fusion protein comprising the test proteinand a fluorescent protein having a multimerization ability; and

(c) selecting the test protein as an association-inducing protein if afluorescent focus is not detected in step (a) but a fluorescent focus isdetected in step (b).

The “test protein” according to the present invention is notparticularly limited, and it is possible to use desired proteinsintended for detection of association-inducing ability.

Note that the “mAG1” (monomeric Azami Green 1) is typically a proteinhaving the amino acid sequence of SEQ ID NO: 135. Moreover, the aminoacid sequence of a protein may be mutated naturally (i.e.,non-artificially). Further, a mutation can also be introducedartificially. Such a mutant can also be used in the present invention,as long as it can emit fluorescence and can be present in a monomericform in the cell.

The fluorescent protein having a multimerization ability used in thescreening method for an association-inducing protein of the presentinvention is as described above.

In the “fusion protein comprising the test protein and mAG1” or the“fusion protein comprising the test protein and the fluorescent proteinhaving a multimerization ability” according to the present invention,the mAG1 or the fluorescent protein having a multimerization ability maybe fused on either the N-terminus side or the C-terminus side of thetest protein. Moreover, the mAG1 or the fluorescent protein having amultimerization ability may be fused to the test protein directly, ormay be fused on indirectly via the spacer protein. The “test protein”according to the present invention may be fused to the above-describedother functional protein. In this case, the other functional protein maybe fused on one or both of the N-terminus side and the C-terminus sideof the fusion protein, or may be fused directly or indirectly betweenthe mAG1 or the fluorescent protein having a multimerization ability andthe test protein.

In the screening of the present invention, the test protein is selectedas an association-inducing protein if a fluorescent focus is notdetected when fused to the mAG1 and expressed in the cell but afluorescent focus is detected when fused to the fluorescent proteinhaving a multimerization ability and expressed in the cell.

<Method for Obtaining Temporal Information and the Like onProtein-Protein Interaction>

As described in Examples, particularly Example 12, later, the method ofthe present invention is capable of detecting not only a protein-proteininteraction taking place, but also a protein-protein interaction ending,on the basis of the presence or absence of the fluorescent focusaccording to the present invention. Moreover, as described in Examplessuch as Examples 19, 24 to 28, it is also possible to trace occurrenceor the like of such a protein-protein interaction over time. Further, asdescribed in Examples such as Examples 20 to 22, the present inventionis also capable of detecting a protein-protein interaction in any regionin a cell without being influenced by localization of anassociation-inducing protein and a fluorescent protein having amultimerization ability, and so forth.

Thus, the present invention can provide a method, wherein thefluorescent focus according to the present invention is detected todetect an interaction taking place or ending, a period until theinteraction takes place or ends, or a duration of the interaction.

In detecting the “interaction taking place or ending” in the mannerdescribed above, the present invention is also capable of specifying anintracellular region where the protein-protein interaction takes placeas described particularly in Example 21 later.

Additionally, as described in Examples such as Examples 19, 23, 27 to 29later, according to the present invention, detecting the “interactiontaking place or ending” makes it possible to detect a signaltransduction occurring and ending, in which the protein-proteininteraction is involved, a period until the signal transduction occursor ends, and a duration of the signal transduction, and also to specifyan intracellular region where the signal transduction occurs.

Moreover, as described in Examples later, the present invention iscapable of detecting the interaction between the first protein and thesecond protein, even if the interaction takes place or ends in responseto a particular stimulus. Thus, the present invention can also provide amethod for detecting the fluorescent focus according to the presentinvention, wherein the fluorescent focus is detected to detect theinteraction taking place or ending in response to a particular stimulus,a period until the interaction takes place or ends, or a duration of theinteraction.

It is only necessary that the “particular stimulus” according to thepresent invention be a stimulus capable of directly or indirectlyinducing or inhibiting a protein-protein interaction. Moreover, the“particular stimulus” may be a stimulus attributable to an endogenousfactor produced in a cell (for example, increase or decrease inintracellular calcium ion concentration, activation or inactivation ofan enzyme), or may be a stimulus applied to a cell from the outside (forexample, administration of a ligand (agonist or antagonist) to areceptor in a cell).

Further, as particularly described in Examples 19, 24 to 28 later, sucha method of the present invention is also capable of detecting aparticular stimulation starting or ending, a period until thestimulation starts or ends, or a duration of the stimulation, bydetecting the fluorescent focus according to the present invention.

Furthermore, as described in Examples such as Examples 11 and 13 later,the method of the present invention is also capable of detecting anincrease or decrease of a protein-protein interaction in accordance witha degree of the particular stimulus (for example, in a case where theparticular stimulus is a drug, its concentration). Thus, in the casewhere the particular stimulus is a drug, the 50% effective concentration(EC50) and the 50% inhibitory concentration (IC50) of the drug against aprotein-protein interaction can be determined by the present invention.

In addition, as described in Example 29 later, the method of the presentinvention is capable of distinguishing and detecting, in a single cell,multiple types of protein-protein interactions, multiple types ofprotein-protein interactions dependent respectively on particularstimuli, and eventually a signal transduction in which theseprotein-protein interactions are involved.

<Screening Method for Protein Interacting with Particular Protein>

As described in Examples, particularly Example 30, later, the presentinvention makes it possible to detect any protein-protein interaction.Thus, the present invention can provide a method for screening for aprotein interacting with a particular protein, wherein

one of the first protein and the second protein is the particularprotein, while the other is a test protein, and

a protein interacting with the particular protein is selected accordingto the detection of the fluorescent focus according to the presentinvention.

The “test protein” according to the present invention is notparticularly limited. Protein groups encoded by cDNA libraries can besuitably used from the viewpoint that it is possible to comprehensivelyand efficiently select proteins interacting with particular proteins.

<Method for Identifying Amino Acid Residue Involved in Protein-ProteinInteraction>

As described in Examples later, the fluorescence intensity of afluorescent focus and a strength of a protein-protein interactioncorrelate with each other in the present invention. Thus, the presentinvention can provide a method for identifying any one of an amino acidresidue in the first protein and an amino acid residue in the secondprotein, which are involved in the protein interaction, wherein

in a case where a protein in which a mutation is introduced is used asany one of the first protein and the second protein, if an intensity ofthe fluorescent focus is reduced in comparison with a case of using aprotein in which no mutation is introduced, the amino acid residue inwhich the mutation is introduced is determined to be involved in theinteraction.

The “fluorescence intensity of the fluorescent focus” according to thepresent invention includes not only a fluorescence intensity of a singlefluorescent focus, but also a total fluorescence intensity offluorescent foci present in a certain region (for example, in one cell,in one field of view and in one fluorescence image observed with afluorescence microscope).

Those skilled in the art can prepare the “protein obtained byintroducing a mutation into the first protein and the like” by selectingknown techniques as appropriate. An example of such known techniquesincludes site-directed mutagenesis.

<Screening Method for Substance Capable of Modulating Protein-ProteinInteraction>

As described above, in the method of the present invention, a strengthof a protein-protein interaction can be grasped on the basis of thefluorescence intensity of the fluorescent focus. Thus, the presentinvention can provide a method comprising the steps of:

expressing in a cell a first fusion protein comprising a first proteinand an association-inducing protein, and a second fusion proteincomprising a second protein and a fluorescent protein having amultimerization ability, in presence of a test compound;

detecting a fluorescent focus formed by an association between the firstfusion protein and the second fusion protein in the cell; and

selecting the test compound as a substance inducing the interaction ifan intensity of the fluorescent focus is higher than an intensity of afluorescent focus formed in absence of the test compound, or selectingthe test compound as a substance suppressing the interaction if theintensity of the fluorescent focus is lower than the intensity of thefluorescent focus formed in the absence of the test compound.

The test compound used in the screening method of the present inventionis not particularly limited. Examples thereof include an expressionproduct from a gene library, a synthetic low-molecular-weight compoundlibrary, a peptide library, an antibody, a substance released by abacterium, a liquid extract and a culture supernatant of cells(microorganisms, plant cells, animal cells), a purified or partiallypurified polypeptide, an extract derived from a marine organism, plant,or animal, soil, and a random phage peptide display library.

Moreover, examples of a state of being in the presence of the testcompound include a state where the cell according to the presentinvention are in contact with the test compound by addition or the likeof the test compound to a medium, and a state where the test compound isintroduced in the cell according to the present invention.

<Kit for Use in Methods of the Present Invention>

The present invention can provide a kit for use in the above-describedmethods. The kit of the present invention is a kit comprising aninstruction and at least one substance selected from the groupconsisting of the following (a) to (j):

(a) a vector comprising a DNA encoding the association-inducing proteinand a cloning site allowing an insertion of a DNA encoding a certainprotein in such a manner that the certain protein is fused to theassociation-inducing protein when expressed;

(b) a vector comprising a DNA encoding the fluorescent protein having amultimerization ability and a cloning site allowing an insertion of aDNA encoding a certain protein in such a manner that the certain proteinis fused to the fluorescent protein when expressed;

(c) a vector comprising a DNA encoding mAG1 and a cloning site allowingan insertion of a DNA encoding a certain protein in such a manner thatthe certain protein is fused to the fluorescent protein when expressed;

(d) a vector encoding the first fusion protein;

(e) a vector encoding the second fusion protein;

(f) a vector set comprising the vector according to any one of (a) and(d) and the vector according to any one of (b) and (e);

(g) a vector set comprising the vector according to (b) and the vectoraccording to (c);

(h) a transformed cell comprising a vector encoding the first fusionprotein;

(i) a transformed cell comprising a vector encoding the second fusionprotein; and

(j) a transformed cell comprising a vector encoding the first fusionprotein and a vector encoding the second fusion protein.

It is only necessary that the vectors according to the present inventioncomprise a regulatory sequence necessary for an expression(transcription and translation) of the inserted DNA in the cellaccording to the present invention. Examples of such a regulatorysequence include a promoter, an enhancer, a silencer, a terminator, apoly(A) tail, and a ribosomal binding site (Shine-Dalgarno (SD)sequence). Further, the vectors according to the present invention maycomprise a selection marker (such as a drug resistance gene), and areporter gene (such as a luciferase gene, a β-galactosidase gene, achloramphenicol acetyltransferase (CAT) gene). Moreover, examples of atype of such vectors according to the present invention include aplasmid vector, an episomal vector, and a viral vector.

The proteins encoded by the vectors according to the present inventionare, as described above, a protein having an association-inducingability, a fluorescent protein having a multimerization ability, mAG1,and fusion proteins with these proteins. From the viewpoint of furtherimproving the efficiency of expressing a DNA encoding such a protein, aDNA having codons optimized in accordance with the species of a cellexpressing the protein (for example, humanized-codon DNA) may beinserted in the vectors according to the present invention.

Examples of the “cloning site allowing an insertion of a DNA encoding acertain protein” in (a), (b), and (c) above include a multiple cloningsite containing one or more restriction-enzyme recognition sites, a TAcloning site, and a GATEWAY (registered trademark) cloning site.

To a preparation of the vectors according to the present invention,other components such as a buffer, a stabilizer, a preservative, and anantiseptic may be added.

The transformed cell according to the present invention can be prepared,as described above, by introducing the vectors according to the presentinvention into a cell. Moreover, to a preparation of the transformedcell according to the present invention, a medium necessary for storageand culturing of the cell and other components such as a stabilizer, apreservative, and an antiseptic may be added or attached.

The “instruction” according to the present invention is an instructionfor utilizing the vectors or the transformed cell in the methods of thepresent invention. The instruction may comprise, for example,experimental techniques and experimental conditions for the methods ofthe present invention, and information on the preparation of the presentinvention (for example, information such as a vector map indicating thebase sequence, cloning site, and the like of the vectors, information onthe origin and nature of the transformed cell, culture conditions of thecell, and so forth).

EXAMPLES

Hereinafter, the present invention will be described more specificallybased on Examples. However, the present invention is not limited to thefollowing Examples.

Example 1 Screening 1 for Association-Inducing Protein

For constructing a system for detecting a protein-protein interaction,proteins were searched for, which functioned as the“association-inducing protein” according to the present invention, onthe basis of the concept illustrated in FIGS. 1 and 2 and by the methodillustrated in FIGS. 3 and 4. Specifically, screened for were proteins,which were present in a dispersed manner in a cell when fused tomonomeric Azami Green 1 (mAG1) (see FIG. 4), while being capable offorming a fluorescent focus (assembly) in a cell when fused to afluorescent protein having a multimerization ability (see FIG. 3).

As such screening targets, first of all, attention was focused on a PB1(Phox and Bemlp) domain. A protein composed of mAG1 fused to a PB1domain of p62 (hereinafter, also referred to as “p62(PB1)”), and aprotein composed of p62(PB1) fused to Azami Green (AG) serving as thefluorescent protein having a multimerization ability were expressed incultured cells. Then, an association between the fusion proteins andeventually the presence or absence of formation of a fluorescent focusformed by the assembly formation were examined by a method describedbelow. Note that AG has been known to forma homotetramer. FIG. 5 showsthe obtained result.

(Preparation of Plasmid DNAs)

As a plasmid DNA for fusing mAG1, phmAG1-MCLinker (manufactured bylimited company Amalgaam Co., Ltd.) was used.

Meanwhile, in preparing a plasmid for fusing AG (phAG-MCLinker), first,a humanized-codon AzamiGreen (AG) gene (DNA encoding a region having theamino acid sequence of SEQ ID NO: 2 (the DNA had the base sequence ofSEQ ID NO: 1) was artificially synthesized. PCR amplification wascarried out using the artificially synthesized humanized-codon AG (hAG)gene as a template, and the following primer set:

hAG forward primer 1; (SEQ ID NO: 57)5′-CTAGCTAGCATTGCCACCATGGTGAGCGTGATCAA GCCCGAG-3′, andhAG reverse primer 1; (SEQ ID NO: 58)5′-ACTACCGGTCTTGGCCTGGCTGGGCAGCATGCTGTA CC-3′.

Then, the amplification product thus obtained was cleaved with NheI andAgeI, and inserted into phmAG1-MCLinker having been treated with thesame restriction enzymes. Thus, phAG-MCLinker was prepared.

Further, in preparing phmAG1-p62(PB1) and phAG-p62(PB1), first, a DNAencoding a PB1 domain of p62 (region having the amino acid sequence ofSEQ ID NO: 4) (the DNA had the base sequence of SEQ ID NO: 3) wasamplified from a cDNA library of HeLaS3 cells by PCR using the followingprimer set:

p62(PB1) forward primer 1; (SEQ ID NO: 59)5′-AAGAATTCGATGGCGTCGCTCACCGTGAAGGCCTACC TTCTGGGC-3′, andp62(PB1) reverse primer 1; (SEQ ID NO: 60)5′-AATTGGCGGCCGCTTATTTCTCTTTAATGTAGATTCGG AAGATGTC-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andNotI, and inserted into phAG-MCLinker and phmAG1-MCLinker having beentreated with the same restriction enzymes; thus, phAG-p62(PB1) andphmAG1-p62(PB1) were prepared, respectively.

(Transfection into Cultured Cells)

HeLaS3 cells were used as cultured cells into which the phAG-p62(PB1)and the phmAG1-p62(PB1) were introduced. Note that HeLaS3 cells werecultured in DMEM Low glucose (manufactured by SIGMA ALDRICH CO.)containing 10% FBS (manufactured by Equitech-Bio Inc.). Moreover, on theday before the transfection, the HeLaS3 cells were seeded onto a 35-mmglass base dish (manufactured by Asahi Glass Co., Ltd.). Further, at thetime of the transfection, 1 μg of the phAG-p62(PB1) or phmAG1-p62(PB1)was diluted with OptiMEM (manufactured by Life TechnologiesCorporation), and 10 μl of PolyFect® Transfection Reagent (manufacturedby QIAGEN N.V.) was added thereto and stirred. Then, the resultant wasfurther mixed with 600 μl of the culture solution, subsequently added tothe HeLaS3 cells, and observed 22 hours thereafter.

(Observation of Transfected Cells)

After the transfection treatment, the HeLaS3 cells were observed in abuffer at pH 7.4 containing Hanks' Balanced Salt Solutions (manufacturedby Life Technologies Corporation) and 20 mM HEPES (manufactured byDojindo Laboratories), using an IX-71 inverted fluorescence microscope(manufactured by Olympus Corporation), a U-MGFPHQ filter (manufacturedby Olympus Corporation), and an ORCA-ER digital camera (manufactured byHamamatsu Photonics K. K.).

As apparent from the result shown in FIG. 5, p62(PB1) was present in adispersed state in the cells when fused to mAG1. On the other hand, whenp62(PB1) was fused to AG, a fluorescent protein having a multimerizationability, fluorescent foci were detected in the cells, revealing thatfusion proteins composed of p62(PB1) and AG were associated with eachother, forming the fluorescent foci. Thus, it was revealed that the PB1domain of p62 itself did not have an association ability, but had anature of forming an assembly (fluorescent focus) when fused to thefluorescent protein having a multimerization ability, suggesting thatthe PB1 domain of p62 was suitably usable as the association-inducingprotein according to the present invention.

Example 2 Detection 1 of Protein-Protein Interaction

In order to verify that the PB1 domain of p62 was suitably usable as theassociation-inducing protein according to the present invention, inother words, to verify that the PB1 domain of p62 was applicable to themodel illustrated in FIGS. 1 and 2, a test was conducted by a methoddescribed below using proteins whose interaction was inducible by addinga drug. FIGS. 6 to 8 show the obtained results.

Note that a FRB domain of a mTOR protein (also referred to as“mTOR(FRB)” or “mTOR(FRB domain)”) and a FKBP12 protein used in Example2 have been known to interact with each other in the presence ofrapamycin (see Chen J et al., Proc Natl Acad Sci USA, May 23, 1995, vol.92, no. 11, pp. 4947 to 4951).

(Preparation of Plasmid DNAs)

In preparing a plasmid for fusing AG (phAG-MNLinker), first, ahumanized-codon Azami Green (AG) gene was amplified by PCR usingphAG-MCLinker as a template and the following primer set:

hAG forward primer 2; (SEQ ID NO: 61)5′-GGACCGGTATGGTGAGCGTGATCAAGCCCGAG-3′, and hAG reverse primer 2;(SEQ ID NO: 62) 5′-TTTCTAGATCACTTGGCCTGGCTGGGCAGCATGC-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into phmAG1-MNLinker (manufactured by limited companyAmalgaam Co., Ltd.) having been treated with the same restrictionenzymes. Thus, phAG-MNLinker was prepared.

Meanwhile, in preparing a plasmid for fusing p62(PB1)(pp62(PB1)-MNLinker), first, the DNA encoding a PB1 domain of p62(region having the amino acid sequence of SEQ ID NO: 4) (the DNA had thebase sequence of SEQ ID NO: 3) was amplified from the cDNA library ofHeLaS3 cells by PCR using the following primer set:

p62(PB1) forward primer 2; (SEQ ID NO: 63)5′-GGGACCGGTATGGCGTCGCTCACCGTGAAGGCCT  ACCTTC-3′, andp62(PB1) reverse primer 2; (SEQ ID NO: 64)5′-ACCTCTAGATTATTTCTCTTTAATGTAGATTCGGA AGATG-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into phmAG1-MNLinker having been treated with thesame restriction enzymes. Thus, pp62(PB1)-MNLinker was prepared.

Further, in preparing the plasmid for fusing p62(PB1)(pp62(PB1)-MCLinker), first, the DNA encoding a PB1 domain of p62(region having the amino acid sequence of SEQ ID NO: 4) (the DNA had thebase sequence of SEQ ID NO: 3) was amplified by PCR using thepp62(PB1)-MNLinker as a template and the following primer set:

p62(PB1) forward primer 3; (SEQ ID NO: 65)5′-TAGCGCTAGCATTGCCACCATGGCGTCGCTCACCGTGAA GGCCTACCTTC-3′, andp62(PB1) reverse primer 3; (SEQ ID NO: 66)5′-AAAACCGGTTTTCTCTTTAATGTAGATTCGGAAGATG-3′.

Then, the amplification product thus obtained was cleaved with NheI andAgeI, and inserted into phmAG1-MCLinker having been treated with thesame restriction enzymes. Thus, pp62(PB1)-MCLinker was prepared.

Moreover, in preparing pmTOR(FRB domain)-hAG, first, a DNA encoding aFRB domain of mTOR (region having the 2025th to 2114th amino acids ofthe mTOR protein, the region had the amino acid sequence of SEQ ID NO:22) (the DNA had the base sequence of SEQ ID NO: 21) was amplified fromthe cDNA library of HeLaS3 cells by PCR using the following primer set:

mTOR(FRB) forward primer; (SEQ ID NO: 67)5′-GCCGAATTCGGCCACCATGGAGATGTGGCATGAAGGCCTG GAAGAGGCATCTCG-3′, andmTOR(FRB) reverse primer; (SEQ ID NO: 68)5′-GGGCTCGAGCCCTGCTTTGAGATTCGTCGGAACACATGATA ATAGAGGTCCC-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andXhoI, and inserted into phAG-MNLinker having been treated with the samerestriction enzymes. Thus, pmTOR(FRB domain)-hAG was prepared. Note thatthe pmTOR(FRB domain)-hAG encodes a fusion protein composed of mTOR(FRB)and an AG protein (the fusion protein may also be referred to as“mTOR(FRB)-AG”).

Further, in preparing pp62(PB1)-FKBP12, first, a DNA encoding FKBP12(full length, region having the amino acid sequence of SEQ ID NO: 24)(the DNA had the base sequence of SEQ ID NO: 23) was amplified from thecDNA library of HeLaS3 cells by PCR using the following primer set:

FKBP12 forward primer; (SEQ ID NO: 69)5′-GCCGAATTCGATGGGAGTGCAGGTGGAAACC-3′, and FKBP12 reverse primer;(SEQ ID NO: 70) 5′-GGGCTCGAGTTATTCCAGTTTTAGAAGCTCCA-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andXhoI, and inserted into pp62(PB1)-MCLinker having been treated with thesame restriction enzymes. Thus, pp62(PB1)-FKBP12 was prepared. Note thatthe pp62(PB1)-FKBP12 encodes a fusion protein composed of p62(PB1) and aFKBP12 protein (the fusion protein may also be referred to as“p62(PB1)-FKBP12”).

Meanwhile, in preparing phAG-FKBP12, first, phAG-MCLinker was cleavedwith NheI and AgeI to prepare a hAG1 gene. Then, the hAG1 gene wasinserted into pp62(PB1)-FKBP12 having been treated with the samerestriction enzymes to cut out a p62(PB1) region therefrom. Thus,phAG-FKBP12 was prepared.

Further, in preparing phmAG1-FKBP12, first, phmAG1-MCLinker was cleavedwith NheI and AgeI to prepare a hmAG1 gene. Then, the hmAG1 gene wasinserted into pp62(PB1)-FKBP12 having been treated with the samerestriction enzymes to cut out a p62(PB1) region therefrom. Thus,phmAG1-FKBP12 was prepared. Note that the phmAG1-FKBP12 encodes a fusionprotein composed of a mAG1 protein and a FKBP12 protein (the fusionprotein may also be referred to as “mAG1-FKBP12”).

Furthermore, in preparing pmTOR(FRB domain)-p62(PB1), first,pp62(PB1)-MNLinker was cleaved with AgeI and XbaI to prepare a p62(PB1)gene. Then, the gene was inserted into pmTOR(FRB domain)-hAG having beentreated with the same restriction enzymes to cut out a hAG regiontherefrom. Thus, pmTOR(FRB domain)-p62(PB1) was prepared. Note that thepmTOR(FRB domain)-p62(PB1) encodes a fusion protein composed ofmTOR(FRB) and p62(PB1) (the fusion protein may also be referred to as“mTOR(FRB)-p62(PB1)”).

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into HeLaS3 cells by the same method asthat described in Example 1:

a combination of pmTOR(FRB domain)-hAG with pp62(PB1)-FKBP12;

a combination of phAG-FKBP12 with pmTOR(FRB domain)-p62(PB1); and

a combination of phmAG1-FKBP12 with pmTOR(FRB domain)-p62(PB1).

Moreover, the transfected cells were observed also by the same method asthat described in Example 1. Then, fluorescence images were capturedbefore 100 nM rapamycin (manufactured by Merck KGaA) was added and 300seconds after the addition.

As apparent from the result shown in FIG. 6, mTOR(FRB)-AG was present ina dispersed manner before rapamycin was added (seethe left panel in FIG.6); meanwhile, fluorescent foci were detected in the cells after theaddition (see the right panel in FIG. 6). Moreover, as apparent from theresult shown in FIG. 7, AG-FKBP12 was present in a dispersed mannerbefore rapamycin was added (see the upper panel in FIG. 7); meanwhile,fluorescent foci were detected in the cells after the addition (see thelower panel in FIG. 7). Thus, it was revealed that by therapamycin-dependent interaction between mTOR(FRB) and the FKBP12protein, mTOR(FRB)-AG was autonomously associated with p62(PB1)-FKBP12,and AG-FKBP12 was autonomously associated with mTOR(FRB)-p62(PB1),thereby both forming fluorescent foci.

On the other hand, as apparent from the result shown in FIG. 8, in thecase where mAG1 was used as the fluorescent protein, no fluorescentfocus was detected in the cells after rapamycin was added, and norapamycin-dependent assembly formation was observed. Thus, it wasrevealed that if the fluorescent protein having a multimerizationability and the association-inducing protein were not used incombination, no assembly was formed by the protein-protein interactionand no fluorescent focus was detected, verifying that the modelillustrated in FIGS. 1 and 2 was practicable.

Example 3 Detection 2 of Protein-Protein Interaction

In order to verify that the PB1 domain of p62 was applicable to themodel illustrated in FIGS. 1 and 2, a test was conducted by a methoddescribed below using the FRB domain of mTOR and FKBP12 described above.FIGS. 9 to 11 show the obtained results.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into HeLaS3 cells by the same method asthat described in Example 2. Then, fluorescence images were capturedbefore 100 nM rapamycin (manufactured by Merck KGaA) was added and 300seconds after the addition.

A combination of pmTOR(FRB domain)-hAG with pp62(PB1)-FKBP12;

a combination of pmTOR(FRB domain)-hmAG1 with pp62(PB1)-FKBP12; and

a combination of pmTOR(FRB domain)-hAG with pp62(PB1_nc)-FKBP12.

(Preparation of Plasmid DNAs)

By the same method as that described in Example 2, pmTOR(FRBdomain)-hmAG1 was prepared by cutting out a DNA encoding hAG frompmTOR(FRB domain)-hAG, and inserted a DNA encoding hmAG1 into thatposition instead.

Moreover, pp62(PB1_nc)-FKBP12 was prepared by introducing a mutationusing pp62(PB1)-FKBP12 as a template, and AMAP™ Multi Site-directedMutagenesis Kit (manufactured by limited company Amalgaam Co., Ltd.)according to the attached instruction, with the following primer:

a primer having the DNA sequence of SEQ ID NO: 154:

5′-GCTTCCAGGCGCACTACCGCGCTGAGCGCGGGGACTTG GTTGCCTTTTC-3′.Note that p62(PB1_nc) is a mutant obtained by introducing a 2-amino acidmutation to an interface where p62(PB1) interacts with another p62(PB1),so that the mutant has no association-inducing ability.

As apparent from the result shown in FIG. 9, similarly to the resultdescribed in Example 2, mTOR(FRB)-AG was present in a dispersed mannerbefore rapamycin was added (see the upper panel in the figure);meanwhile, fluorescent foci were detected in the cells after theaddition (see the lower panel in the figure).

On the other hand, as apparent from the result shown in FIG. 10,similarly to the result described in Example 2, in the case where mAG1was used as the fluorescent protein, no fluorescent focus was detectedin the cells even after rapamycin was added, and no rapamycin-dependentassembly formation was observed.

Further, as apparent from the result shown in FIG. 11, in the case wherethe mutant p62(PB1_nc) no longer capable of forming a homomultimer wasused in place of p62(PB1) also, no fluorescent focus was detected in thecells even after rapamycin was added, and no rapamycin-dependentassembly formation was observed.

Thus, it was revealed that if the fluorescent protein having amultimerization ability and the association-inducing protein were notused in combination, no assembly was formed by the protein-proteininteraction and no fluorescent focus was detected, verifying that themodel illustrated in FIGS. 1 and 2 was practicable.

It should be noted that the method for detecting a protein-proteininteraction of the present invention is a method totally different fromconventional methods for detecting a protein-protein interaction in thatfluorescent foci are autonomously formed only when a protein-proteininteraction takes place.

Example 4 Screening 2 for Association-Inducing Protein

In order to find out association-inducing proteins having naturessimilar to those of p62(PB1), screening was carried out by the samemethod as that described in Example 1.

As such screening targets other than the PB1 domain, attention wasfocused on a SAM domain. A protein composed of mAG1 fused to a PB1domain or a SAM domain derived from proteins, or a protein composed ofAG serving as the fluorescent protein having a multimerization abilityfused to a PB1 domain or a SAM domain derived from proteins wasexpressed in cultured cells. Then, an association between the fusionproteins and eventually the presence or absence of formation of afluorescent focus formed by the association were examined by a methoddescribed below. FIGS. 12 to 15 show the obtained results.

(Preparation of Plasmid DNAs)

To fuse the PB1 domain or the SAM domain derived from the proteins onthe C-terminus side of a fluorescent protein via a flexible linker forthe expression, phmAG1-MCLinker was used as a plasmid for fusing mAG1,and phAG-MCLinker was used as a plasmid for fusing AG.

Specifically, in preparing phmAG1-MEK5(PB1) and phAG-MEK5 (PB1), first,a DNA encoding a PB1 domain of MEK5 (region having the 16th to 109thamino acids of the MEK5 protein, the region had the amino acid sequenceof SEQ ID NO: 6, also referred to as “MEK5(PB1)”) (the DNA had the basesequence of SEQ ID NO: 5) was amplified from the cDNA library of HeLaS3cells by PCR using the following primer set:

MEK5(PB1) forward primer; (SEQ ID NO: 71)5′-CCGAATTCGGTGCTGGTAATTCGCATCAAGATCCCAAA-3′,  andMEK5(PB1) reverse primer; (SEQ ID NO: 72)5′-TTCTCGAGTTAGCAGGCTCTTGGAAATATCTGCAG-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andXhoI, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-MEK5(PB1) andphAG-MEK5(PB1) were prepared, respectively. Note that these plasmid DNAsencode a fusion protein composed of a mAG1 protein and MEK5(PB1) (thefusion protein may also be referred to as “mAG1-MEK5(PB1)”), and afusion protein composed of an AG protein and MEK5 (PB1) (the fusionprotein may also be referred to as “AG-MEK5(PB1)”), respectively.

Meanwhile, in preparing phmAG1-Nbr1(PB1) and phAG-Nbr1(PB1), first, aDNA encoding a PB1 domain of Nbr1 (region having the 4th to 85th aminoacids of the Nbr1 protein, the region had the amino acid sequence of SEQID NO: 8, also referred to as “Nbr1(PB1)”) (the DNA had the basesequence of SEQ ID NO: 7) was amplified from the cDNA library of HeLaS3cells by PCR using the following primer set:

Nbr1(PB1) forward primer; (SEQ ID NO: 73)5′-AAGAATTCGGCAGGTTACTCTAAATGTGACTTTTAAA-3′, andNbr1(PB1) reverse primer; (SEQ ID NO: 74)5′-TTCTCGAGTTACCCTTCGTGGACTTGCATCTGCAGTT-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andXhoI, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-Nbr1(PB1) andphAG-Nbr1(PB1) were prepared, respectively. Note that these plasmid DNAsencode a fusion protein composed of a mAG1 protein and Nbr1 (PB1) (thefusion protein may also be referred to as “mAG1-Nbr1(PB1)”), and afusion protein composed of an AG protein and Nbr1(PB1) (the fusionprotein may also be referred to as “AG-Nbr1(PB1)”), respectively.

Further, in preparing phmAG1-PKCiota(PB1) and phAG-PKCiota(PB1), first,a DNA encoding a PB1 domain of PKCiota (region having the 16th to 99thamino acids of the PKCiota protein, the region had the amino acidsequence of SEQ ID NO: 10, also referred to as “PKCiota(PB1)”) (the DNAhad the base sequence of SEQ ID NO: 9) was amplified from the cDNAlibrary of HeLaS3 cells by PCR using the following primer set:

PKCiota (PB1) forward primer; (SEQ ID NO: 75)5′-AAGAATTCGCAGGTCCGGGTGAAAGCCTACTACCGCG-3′, andPKCiota (PB1) reverse primer 18; (SEQ ID NO: 76)5′-TTCTCGAGTTAACAAGGGAACACATGAATCAAGAGTTCAG-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andXhoI, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-PKCiota(PB1) andphAG-PKCiota(PB1) were prepared, respectively. Note that these plasmidDNAs encode a fusion protein composed of a mAG1 protein and PKCiota(PB1)(the fusion protein may also be referred to as “mAG1-PKCiota(PB1)”), anda fusion protein composed of an AG protein and PKCiota(PB1) (the fusionprotein may also be referred to as “AG-PKCiota(PB1)”), respectively.

Moreover, in preparing phmAG1-TFG(PB1) and phAG-TFG(PB1), first, a DNAencoding a PB1 domain of TFG (region having the 10th to 91st amino acidsof the TFG protein, the region had the amino acid sequence of SEQ ID NO:12, also referred to as “TFG(PB1)”) (the DNA had the base sequence ofSEQ ID NO: 11) was amplified from the cDNA library of HeLaS3 cells byPCR using the following primer set:

TFG (PB1) forward primer 1; (SEQ ID NO: 77)5′-AACTGCAGCAAAGCTAATCATCAAAGCTCAACTTGGGGA-3′, andTFG (PB1) reverse primer 1; (SEQ ID NO: 78)5′-TTAAGCTTTTAATTAACAAATAATGTCAGTTTCAGTAT-3′.

Then, the amplification product thus obtained was cleaved with PstI andHindIII, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-TFG(PB1) andphAG-TFG(PB1) were prepared, respectively. Note that these plasmid DNAsencode a fusion protein composed of a mAG1 protein and TFG(PB1) (thefusion protein may also be referred to as “mAG1-TFG(PB1)”), and a fusionprotein composed of an AG protein and TFG(PB1) (the fusion protein mayalso be referred to as “AG-TFG(PB1)”), respectively.

Further, in preparing phmAG1-TEL(SAM) and phAG-TEL(SAM), first, a DNAencoding a SAM domain of TEL (region having the 38th to 124th aminoacids of the TFG protein, the region had the amino acid sequence of SEQID NO: 14, also referred to as “TEL(SAM)”) (the DNA had the basesequence of SEQ ID NO: 13) was amplified from the cDNA library of HeLaS3cells by PCR using the following primer set:

TEL (SAM) forward primer; (SEQ ID NO: 79)5′-AAAAGGATCCGCCACCATGCCTCGAGCGCTCAGGATGGAGGAA-3′, andTEL (SAM) reverse primer; (SEQ ID NO: 80)5′-AAAAAAGCTTTTACCTCTGCTTCAGAATATGCTGAAGGAGTT-3′.

Then, the amplification product thus obtained was cleaved with BamHI andHindIII, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-TEL(SAM) andphAG-TEL(SAM) were prepared, respectively. Note that these plasmid DNAsencode a fusion protein composed of a mAG1 protein and TEL(SAM) (thefusion protein may also be referred to as “mAG1-TEL(SAM)”), and a fusionprotein composed of an AG protein and TEL(SAM) (the fusion protein mayalso be referred to as “AG-TEL(SAM)”), respectively.

Additionally, in preparing phmAG1-EphB2(SAM) and phAG-EphB2 (SAM),first, a DNA encoding a SAM domain of EphB2 (region having the 905th to981st amino acids of the EphB2 protein, the region had the amino acidsequence of SEQ ID NO: 16, also referred to as “EphB2 (SAM)”) (the DNAhad the base sequence of SEQ ID NO: 15) was amplified from the cDNAlibrary of HeLaS3 cells by PCR using the following primer set:

EphB2 (SAM) forward primer; (SEQ ID NO: 81)5′-AAAAGGATCCGCCACCATGCTGGACCGCACGATCCCCGA-3′, andEphB2 (SAM) reverse primer; (SEQ ID NO: 82)5′-AAAAAAGCTTTTAAATCTGGTTCATCTGCGCCCG-3′.

Then, the amplification product thus obtained was cleaved with BamHI andHindIII, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-EphB2 (SAM) andphAG-EphB2(SAM) were prepared, respectively. Note that these plasmidDNAs encode a fusion protein composed of a mAG1 protein and EphB2(SAM)(the fusion protein may also be referred to as “mAG1-EphB2(SAM)”), and afusion protein composed of an AG protein and EphB2(SAM) (the fusionprotein may also be referred to as “AG-EphB2(SAM)”), respectively.

Furthermore, in preparing phmAG1-DGK delta (SAM) and phAG-DGKdelta(SAM), first, a DNA encoding a SAM domain of DGK delta (regionhaving the 1097th to 1164th amino acids of the DGK delta protein, theregion had the amino acid sequence of SEQ ID NO: 18, also referred to as“DGK delta(SAM)”) (the DNA had the base sequence of SEQ ID NO: 17) wasamplified from the cDNA library of HeLaS3 cells by PCR using thefollowing primer set:

DGK delta (SAM) forward primer; (SEQ ID NO: 83)5′-AAAAGGTACCGCCACCATGCCGGTTCACCTCTGGGGGACA-3′, andDGK delta (SAM) reverse primer; (SEQ ID NO: 84)5′-AAAAAAGCTTTTAGCTGCGGCTCAGCTCCTTGAT-3′.

Then, the amplification product thus obtained was cleaved with KpnI andHindIII, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-DGK delta(SAM)and phAG-DGK delta(SAM) were prepared, respectively. Note that theseplasmid DNAs encode a fusion protein composed of a mAG1 protein and DGKdelta(SAM) (the fusion protein may also be referred to as “mAG1-DGKdelta(SAM)”), and a fusion protein composed of an AG protein and DGKdelta (SAM) (the fusion protein may also be referred to as “AG-DGKdelta(SAM)”), respectively.

In addition, in preparing phmAG1-Tankyrase(SAM) and phAG-Tankyrase(SAM),first, a DNA encoding a SAM domain of Tankyrase (region having the 952ndto 1078th amino acids of the Tankyrase protein, the region had the aminoacid sequence of SEQ ID NO: 20, also referred to as “Tankyrase(SAM)”)(the DNA had the base sequence of SEQ ID NO: 19) was amplified from thecDNA library of HeLaS3 cells by PCR using the following primer set:

Tankyrase (SAM) forward primer; (SEQ ID NO: 85)5′-AAAAGGATCCGCCACCATGCTGATAGATGCCATGCCCCCAGA-3′, andTankyrase (SAM) reverse primer; (SEQ ID NO: 86)5′-AAAAAAGCTTTTAAATTCGAATGACATTGTATCTGTTGAAGA-3′.

Then, the amplification product thus obtained was cleaved with BamHI andHindIII, and inserted into phmAG1-MCLinker and phAG-MCLinker having beentreated with the same restriction enzymes; thus, phmAG1-Tankyrase(SAM)and phAG-Tankyrase(SAM) were prepared, respectively. Note that theseplasmid DNAs encode a fusion protein composed of a mAG1 protein andTankyrase(SAM) (the fusion protein may also be referred to as“mAG1-Tankyrase(SAM)”), and a fusion protein composed of an AG proteinand Tankyrase(SAM) (the fusion protein may also be referred to as“AG-Tankyrase(SAM)”), respectively.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the plasmid DNAs was introduced into HeLaS3 cells by the samemethod as that described in Example 1. Moreover, the transfected cellswere observed also by the same method as that described in Example 1.

As apparent from the results shown in FIGS. 13, 14, and 15,PKCiota(PB1), TFG(PB1), TEL(SAM), DGK delta(SAM), and Tankyrase(SAM)were dispersed when fused to mAG1, and formed fluorescent foci(assemblies) when fused to AG. Note that the cells expressingAG-PKCiota(PB1) included both of cells forming fluorescent foci andcells not forming fluorescent foci. In contrast, in all the cellsexpressing the other fusion proteins comprising the AG protein,fluorescent foci were detected.

On the other hand, as apparent from the results shown in FIGS. 12 and15, when MEK5(PB1), Nbr1(PB1), and EphB2(SAM) were fused to mAG1 or whenfused to AG, no fluorescent focus formation was observed.

Thus, it was revealed that TFG(PB1), TEL(SAM), DGK delta(SAM), andTankyrase(SAM) were usable as the association-inducing protein accordingto the present invention.

Example 5 Detection 3 of Protein-Protein Interaction

In order to verify that the association-inducing proteins selected inExample 4 were suitably usable in the method for detecting aprotein-protein interaction of the present invention, a test describedbelow was conducted by the same method as that described in Example 2.FIG. 16 shows the obtained result.

(Preparation of Plasmid DNAs)

In preparing pFKBP12-TFG(PB1), first, a TFG(PB1) gene was amplified fromphAG-TFG (PB1) by PCR using the following primer set:

TFG (PB1) forward primer 2; (SEQ ID NO: 87)5′-AAACCGGTAAGCTAATCATCAAAGCTCAACTT-3′, and TFG (PB1) reverse primer 2;(SEQ ID NO: 88) 5′-TTTCTAGATTAATTAACAAATAATGTCAGTTTCAGTAT-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into pFKBP12-p62(PB1) having been treated with thesame restriction enzymes to cut out a p62(PB1) region therefrom. Thus,pFKBP12-TFG(PB1) was prepared. Note that the pFKBP12-TFG(PB1) encodes afusion protein composed of a FKBP12 protein and TFG(PB1) (the fusionprotein may also be referred to as “FKBP12-TFG(PB1)”).

Meanwhile, in preparing pFKBP12-TEL(SAM), first, a TEL (SAM) gene wasamplified from phAG-TEL (SAM) by PCR using the following primer set:

TEL (SAM) forward primer 2; (SEQ ID NO: 89)5′-AAAAACCGGTCCTCGAGCGCTCAGGATGGAGGAA-3′, andTEL (SAM) reverse primer 2; (SEQ ID NO: 90)5′-AAAATCTAGATTACCTCTGCTTCAGAATATGCTGAAGGAGTT-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into pFKBP12-p62(PB1) having been treated with thesame restriction enzymes to cut out a p62(PB1) region therefrom. Thus,pFKBP12-TEL(SAM) was prepared. Note that the pFKBP12-TEL(SAM) encodes afusion protein composed of a FKBP12 protein and TEL(SAM) (the fusionprotein may also be referred to as “FKBP12-TEL(SAM)”).

Further, in preparing pFKBP12-DGK delta(SAM), first, a DGK delta(SAM)gene was amplified from phAG-DGK delta(SAM) by PCR using the followingprimer set:

DGK delta (SAM) forward primer 2; (SEQ ID NO: 91)5′-AAAAACCGGTCCGGTTCACCTCTGGGGGACAGA-3′, andDGK delta (SAM) reverse primer 2; (SEQ ID NO: 92)5′-AAAATCTAGATTAGCTGCGGCTCAGCTCCTTGAT-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into pFKBP12-p62(PB1) having been treated with thesame restriction enzymes to cut out a p62(PB1) region therefrom. Thus,pFKBP12-DGK delta(SAM) was prepared. Note that the pFKBP12-DGKdelta(SAM) encodes a fusion protein composed of a FKBP12 protein and DGKdelta (SAM) (the fusion protein may also be referred to as“FKBP12-DGKd(PB1)”).

Moreover, in preparing pFKBP12-Tankyrase(SAM), first, a Tankyrase(SAM)gene was amplified from phAG-Tankyrase(SAM) by PCR using the followingprimer set:

Tankyrase (SAM) forward primer 2; (SEQ ID NO: 93)5′-AAAAACCGGTCTGATAGATGCCATGCCCCCAGA-3′, andTankyrase (SAM) reverse primer 2; (SEQ ID NO: 94)5′-AAAATCTAGATTAAATTCGAATGACATTGTATCTGTTGAAGA-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into pFKBP12-p62(PB1) having been treated with thesame restriction enzymes to cut out a p62(FBI) region therefrom. Thus,pFKBP12-Tankyrase(SAM) was prepared. Note that pFKBP12-Tankyrase(SAM)encodes a fusion protein composed of a FKBP12 protein and Tankyrase(SAM) (the fusion protein may also be referred to as“FKBP12-Tankyrase(SAM)”).

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into HeLaS3 cells by the same method asthat described in Example 1:

a combination of pmTOR(FRB domain)-hAG with pFKBP12-TFG(PB1);

a combination of pmTOR(FRB domain)-hAG with pFKBP12-TEL(SAM);

a combination of pmTOR(FRB domain)-hAG with pFKBP12-DGK delta(SAM); and

a combination of pmTOR(FRB domain)-hAG with pFKBP12-Tankyrase(SAM).

Moreover, the transfected cells were observed also by the same method asthat described in Example 1. Then, it was confirmed that no fluorescentfocus was detected in each of the cultured cells before 100 nM rapamycin(manufactured by Merck KGaA) was added, and fluorescence images werecaptured 300 seconds after the addition.

As apparent from the result shown in FIG. 16, even when any of thefusion proteins were expressed, mTOR(FRB domain)-AG was present in adispersed manner before rapamycin was added (unillustrated); meanwhile,fluorescent foci were detected in all the cells after the addition. Inother words, it was revealed that by the rapamycin-dependent interactionbetween mTOR(FRB domain) and the FKBP12 protein, mTOR(FRB domain)-AG wasautonomously associated with FKBP12-TEL(SAM) and so forth, therebyforming fluorescent foci.

Thus, it was revealed that the use of not only p62(PB1) but also theother types of domains derived from the other proteins, such asTEL(SAM), as the association-inducing protein enabled detection of aprotein-protein interaction. Moreover, this result verified that themodel illustrated in FIGS. 3 and 4 was practicable as the screeningmethod for an association-inducing protein according to the presentinvention.

Example 6 Detection 3 of Protein-Protein Interaction

Whether or not fluorescent proteins other than the AG protein wereapplicable as the fluorescent protein having a multimerization abilityin the method for detecting a protein interaction of the presentinvention was examined by a method described below. FIG. 17 shows theobtained result.

Note that the fluorescent proteins thus examined were Kusabira-Orange 1(KO1), dimeric Keima-Red (dKeima), and Kikume Green-Red (KikGR), whichhave been known to form a homodimer, a homodimer, and a homotetramer,respectively.

(Preparation of Plasmid DNAs)

In preparing pmTOR(FRB domain)-hKO1, first, a hKO1 gene was amplifiedfrom phKO1-MN1 (manufactured by limited company Amalgaam Co., Ltd.) byPCR using the following primer set:

hKO1 forward primer; (SEQ ID NO: 95)5′-AAAAACCGGTATGGTGAGCGTGATCAAGCCCGAG-3′, and hKO1 reverse primer;(SEQ ID NO: 96) 5′-AAAATCTAGATTAGCAGTGGGCCACGGCGTCCTCC-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into pmTOR(FRB domain)-hAG having been treated withthe same restriction enzymes to cut out a hAG region therefrom. Thus,pmTOR(FRB domain)-hKO1 was prepared.

Meanwhile, in preparing pmTOR(FRB domain)-hdKeima-Red, first, a hdKeimagene was prepared by cleaving phdKeima-Red-MNLinker (manufactured bylimited company Amalgaam Co., Ltd.) with AgeI and XbaI. Then, theobtained hdKeima gene was inserted into pmTOR(FRB domain)-hAG havingbeen treated with the same restriction enzymes to cut out a hAG regiontherefrom. Thus, pmTOR(FRB domain)-hdKeima-Red was prepared.

Further, in preparing pmTOR(FRB domain)-hKikGR1, first, a hKikGR1 genewas prepared by cleaving phKikGR1-MNLinker (manufactured by limitedcompany Amalgaam Co., Ltd.) with AgeI and XbaI. Then, the obtainedhKikGR1 gene was inserted into pmTOR(FRB domain)-hAG having been treatedwith the same restriction enzymes to cut out a hAG region therefrom.Thus, pmTOR(FRB domain)-phKikGR1 was prepared.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into HeLaS3 cells by the same method asthat described in Example 1:

a combination of pmTOR(FRB domain)-hKO1 with pp62(PB1)-FKBP12;

a combination of pmTOR(FRB domain)-hdKeima-Red with pp62(PB1)-FKBP12;

a combination of pmTOR(FRB domain)-hKikGR1 with pp62(PB1)-FKBP12; and

a combination of pmTOR(FRB domain)-hAG with pp62(PB1)-FKBP12.

Moreover, the transfected cells were observed also by the same method asthat described in Example 1. Note that the KO1 was observed using anexcitation filter (BP520-540HQ, Olympus Corporation), a dichroic mirror(DM545HQ, manufactured by Olympus Corporation), and an absorption filter(BA555-600HQ, manufactured by Olympus Corporation). The dKeima wasobserved using an excitation filter (440AF21, manufactured by OMEGAOPTICAL, INC.), a dichroic mirror (455DRLP, manufactured by OMEGAOPTICAL, INC.), and an absorption filter (610ALP, manufactured by OMEGAOPTICAL, INC.). Then, fluorescence images were captured before 100 nMrapamycin (manufactured by Merck KGaA) was added and 300 seconds afterthe addition.

As apparent from the result shown in FIG. 17, as in the case of usingthe AG protein, all of the examined fluorescent proteins demonstratedthat mTOR(FRB) fused to the fluorescent proteins was present in adispersed manner before rapamycin was added; meanwhile, fluorescent foci(assemblies) were formed after the addition. Thus, it was verified that,in the present invention, the use of not only the AG protein but alsothe other fluorescent proteins having a multimerization ability, such asthe KO1 protein, enabled detection of a protein-protein interaction.

Example 7 Screening 1 of Fluorescent Protein Having MultimerizationAbility

Whether or not a fluorescent protein other than AG, KO1, dKeima, andKikGR was applicable as the fluorescent protein having a multimerizationability in the method for detecting a protein interaction of the presentinvention was examined by a method described below. Specifically,examined was whether or not an assembly (fluorescent focus) as shown inFIG. 3 was formed when monomeric Kusabira-Orange (mKO2) was fused withp62(PB1) serving as the association-inducing protein and expressed incells. FIG. 18 shows the obtained result.

First, as in the case of “phmAG1-p62(PB1) and phAG-p62(PB1)” describedin Example 1, the DNA encoding p62(PB1) was amplified by PCR. Theamplification product thus obtained was cleaved with EcoRI and NotI, andinserted into phmKO2-MCLinker (manufactured by limited company AmalgaamCo., Ltd.) having been treated with the same restriction enzymes. Thus,phmKO2-p62(PB1) was prepared. Then, the phmKO2-p62(PB1) was introducedinto HeLaS3 cells by the same method as that described in Example 1.Moreover, the transfected cells were observed also by the same method asthat described in Example 6. FIG. 18 shows the obtained result.

As apparent from the result shown in FIG. 18, mKO2-p62(PB1) proteinsencoded by phmKO2-p62(PB1) were associated with each other, therebyforming fluorescent foci. Thus, it was revealed that not only theabove-described fluorescent proteins capable of forming a homomultimerin cells, such as AG, KO1, dKeima, and KikGR, but also mKO2 generallybelieved to be a monomeric fluorescent protein were usable as thefluorescent protein having a multimerization ability in the method ofthe present invention.

Example 8 Screening 2 of Fluorescent Proteins Having MultimerizationAbility

It was confirmed by the following method that other than AG, KO1,dKeima, and KikGR described above, MiCy1, KCy1, dAG (AB), and dAG (AC)(fluorescent protein capable of homodimerization) as well as TGuv,Momiji, COR3.01, COR5, and DsRed2 (fluorescent protein capable ofhomotetramerization), which had been known as fluorescent proteinscapable of forming a homomultimer in cells, were also usable in themethod for detecting a protein interaction of the present invention.

Moreover, in order to find out fluorescent proteins generally believedto be monomeric fluorescent proteins such as mKO2 but usable in themethod of the present invention, screening was carried out by thefollowing method.

(Method for Detecting Protein-Protein Interaction)

By the same method as that described in Examples 5 and 6,p62(PB1)-FKBP12, FKBP12-DGKd(SAM), FKBP12-TEL(SAM), orFKBP12-Tankyrase(SAM), and mTOR(FRB) fused to corresponding one offluorescent proteins shown in Table 1 were expressed in HeLaS3 cells toevaluate a degree of fluorescent focus (assembly) formation afterrapamycin was added. Note that plasmid DNAs encoding fusion proteinscomposed of mTOR(FRB) and the corresponding fluorescent proteins wereprepared as appropriate by the same method as that described in Example2. Moreover, regarding the combinations of FKBP12-Tankyrase(SAM) withmKO2, mKeima, mMiCy1, mKO1, MiCy1, and TGuv, the test was conducted byintroducing the genes into 293T cells. Specifically, the 293T cells werecultured in DMEM High glucose (manufactured by SIGMA ALDRICH CO.)containing 10% FBS (manufactured by Equitech-Bio Inc.). Moreover, the293T cells were seeded onto an 8-well chamber (manufactured by Nunc A/S)6 hours before the plasmid DNAs were introduced. Further, at the time ofthe transfection, 200 ng of the plasmid DNA encoding FKBP12-Tankyrase(SAM) and 200 ng of one of the plasmid DNAs encoding fusion proteinscomposed of the fluorescent proteins and mTOR(FRB) were diluted with 30μl of OptiMEM (manufactured by Life Technologies Corporation), and 1.2μl of TurboFect Transfection Reagent (manufactured by Thermo FisherScientific Inc.) was added thereto and stirred. Then, the resultant wasfurther mixed with 300 μl of the culture solution, subsequently added tothe 293T cells, and observed 48 hours later. Table 1 shows the obtainedresult. In Table 1, “+++” indicates a combination from which fluorescentfoci were observed in 50% or more of the HeLaS3 cells; “++” indicates acombination from which fluorescent foci were observed in 50% or less ofthe HeLaS3 cells; and “+” indicates a combination from which fluorescentfoci were observed in 293T cells expressing a larger amount of proteinsthan that in the HeLaS3 cells.

TABLE 1 P62 DGKd TEL Tankyrase (PB1) (SAM) (SAM) (SAM) mKO2 monomer ++++++ ++ + mKeima monomer +++ +++ ++ + mMiCy1 monomer +++ +++ +++ + mKO1monomer +++ +++ +++ + mKikGR1 monomer +++ +++ +++ +++ MiCy1 dimer ++++++ +++ + KCy1 dimer +++ +++ +++ ++ KO1 dimer +++ +++ +++ ++ dKeimadimer +++ +++ +++ ++ dAG(AB) dimer +++ +++ +++ ++ dAG(AC) dimer +++ ++++++ ++ TGuv tetramer +++ +++ +++ + Momiji tetramer +++ +++ +++ +++ KikGRtetramer +++ +++ +++ +++ AG tetramer +++ +++ +++ +++ COR3.01 tetramer+++ +++ +++ +++ COR5 tetramer +++ +++ +++ +++ DsRed2 tetramer +++ ++++++ +++

As apparent from the result shown in Table 1, it was confirmed that allof the fluorescent proteins capable of forming a homomultimer in cellswere usable in the method of the present invention. Moreover, theresults of TEL (SAM) and Tankyrase (SAM) seem to suggest a tendency thatthe higher the multimerization ability of a fluorescent protein, themore likely that fluorescent foci (assemblies) are formed by theprotein-protein interaction. Further, as shown in Table 1, it wasrevealed that mKeima, mMiCy1, mKO1, and mKikGR1 generally believed to bemonomeric fluorescent proteins, other than mKO2, were also usable in themethod of the present invention.

Example 9 Detection 5 of Protein-Protein Interaction

It was confirmed by a method described below that, even in combinationwith TFG(PB1) serving as the association-inducing protein, the use ofthe fluorescent proteins having a multimerization ability and confirmedin Examples 6 to 8 to be usable in the method of the present inventionenabled detection of a protein-protein interaction. Table 2 shows theobtained result.

(Method for Detecting Protein-Protein Interaction)

By the same method as that described in Examples 5 and 6, TFG(PB1) andmTOR(FRB) fused to mKikGR1, dAG(AC), Momiji, KikGR, AG, COR3.01, COR5,or DsRed2 were expressed in HeLaS3 cells to evaluate a degree offluorescent focus (assembly) formation after rapamycin was added.Moreover, by the same method as that described in Example 8, TFG(PB1)and mTOR(FRB) fused to KO1 or dAG (AB) were expressed in 293T cells toevaluate a degree of fluorescent focus (assembly) formation afterrapamycin was added. Table 2 shows the obtained result. In Table 2, “++”indicates a combination from which fluorescent foci were observed in 50%or less of the HeLaS3 cells, and “+” indicates a combination from whichfluorescent foci were observed in the 293T cells expressing a largeramount of proteins than that in the HeLaS3 cells.

TABLE 2 TFG(PB1) mKikGR1 monomer ++ KO1 dimer + dAG(AB) dimer + dAG(AC)dimer ++ Momiji tetramer ++ KikGR tetramer ++ AG tetramer ++ COR3.01tetramer ++ COR5 tetramer ++ DsRed2 tetramer ++

As apparent from the result shown in Table 2, it was confirmed that theuse of the fluorescent proteins having a multimerization ability incombination with the other association-inducing protein than theproteins described in Example 8 also enabled detection of aprotein-protein interaction.

Example 10 Detection 6 of Protein-Protein Interaction

The fluorescent focus (assembly) according to the present invention is,as described above, attributable to a protein-protein interaction.Hence, a fluorescence intensity of a fluorescent focus presumablyreflects a strength of the protein-protein interaction. Moreover, in themethod for detecting a protein-protein interaction, quantification andcomparison, if possible, of the strength of the interaction are usefulin evaluating a substance (inhibitor) suppressing a protein-proteininteraction, and evaluating a factor modulating a protein-proteininteraction.

For this reason, the concentration of a compound inducing aprotein-protein interaction was changed to test whether or not thefluorescence intensity of fluorescent foci (assemblies) was changed in amanner dependent on the concentration of the compound by a methoddescribed below.

(Transfection into Cells, and Observation and Analysis of TransfectedCells)

pmTOR(FRB domain)-hAG and pFKBP12-p62(PB1) were mixed in equal amountsand introduced into HeLaS3 cells by the same method as that described inExample 1. The cells were collected 24 hours after the introduction, andseeded onto 96 MicroWell Optical Bottom Plate (manufactured by Nunc A/S)at 20000 cells/well. Then, 24 hours after the seeding, a solution ofHoechst 33342 (manufactured by Dojindo Laboratories) diluted with anobservation buffer to 5.6 μg/ml was added to the plate, and allowed forfurther culturing for 30 minutes. Thereafter, the plate was washed twicewith D-PBS(−) (manufactured by Wako Pure Chemical Industries, Ltd.).Then, the medium was replaced with rapamycin having been diluted with anobservation buffer to predetermined concentrations, and 15 minutes laterthe resultant was fixed with 4% Paraformaldehyde Phosphate BufferSolution (manufactured by Wako Pure Chemical Industries, Ltd.). Notethat the concentrations of rapamycin used were 0.1 nM, 0.2 nM, 0.5 nM,1.4 nM, 4.1 nM, 12.3 nM, 37.0 nM, 111.1 nM, 333.3 nM, and 1000 nM. Then,the prepared samples were observed using IN Cell Analyzer 1000(manufactured by GE Healthcare). FIG. 19 shows part of the obtainedresult.

Further, fluorescence images were analyzed in multiple fields of view. Atotal luminance (total fluorescence intensity) of fluorescent foci(assemblies) per cell in an image of wells to which rapamycin was addedat predetermined concentrations was calculated to analyze a correlationwith the rapamycin concentration. FIG. 20 shows part of the obtainedresult. Note that, in FIG. 20, the X axis represents the concentrationof rapamycin added to each well; the Y axis represents the totalluminance (total fluorescence intensity) of fluorescent foci(assemblies) per cell; and dots represent the measurement values.Moreover, using Igor® (manufactured by WaveMetrics, Inc.), a fittingcurve was drawn through the dots, which represents a function fitted toan equation: y=base+(max−base)/[1+(xhalf/x)^rate], where [DotIntensity/Cells]=y, and [Canc. (nM)]=x. (base=0.0028731, max=0.1823,rate=1.4516, xhalf=46.99).

As apparent from the results shown in FIGS. 19 and 20, it was revealedthat when the compound rapamycin inducing an interaction betweenmTOR(FRB domain) and a FKBP12 protein was added to the cells expressingmTOR(FRB domain)-AG and FKBP12-p62(PB1), the fluorescence intensity offluorescent foci was increased in a manner dependent on theconcentration of rapamycin added. It was also demonstrated that theassemblies between mTOR(FRB domain)-AG and FKBP12-p62(PB1) were formedin a manner dependent on the concentration.

Example 11 Detection 7 of Protein-Protein Interaction

Whether or not the method of the present invention was utilizable indetermining the 50% effective concentration (EC50) and the 50%inhibitory concentration (IC50) of a drug against a protein-proteininteraction was evaluated by a method described below.

(Preparation of Plasmid DNA)

Using pmTOR(FRB domain)-hAG described in Example 2 and pFucci-S/G2/MGreen-Hyg (manufactured by limited company Amalgaam Co., Ltd.),pmTOR(FRB domain)-hAG_Hyg was prepared according to a conventionalmethod, so that the drug resistance gene was converted to a hygromycin Bresistance gene.

(Preparation of Stably-Expressing Cell Line)

By the same method as that described in Example 1, the pmTOR(FRBdomain)-hAG_Hyg and pp62(PB1)-FKBP12 described in Example 2 wereintroduced in a HeLaS3 cell line, and cultured.

Moreover, 24 hours after the plasmid DNAs were introduced in the HeLaS3cell line, the medium was replaced with a medium containing 600 μg/mL ofG418 (manufactured by Wako Pure Chemical Industries, Ltd.) and 150 μg/mLof hygromycin (manufactured by Nacalai Tesque, Inc.). Then, cellssurvived after culturing for one week with this medium were cloned by acolony pick-up method.

(Observation and Analysis of Transfected Cells)

The cloned cell lines were seeded onto 96-well plates. Then, afterwashing twice with PBS on the next day, an observation buffer containingHoechst 33342 (manufactured by Dojindo Laboratories) was added to thecell line seeded onto each well, followed by incubation at 37° C. for 15minutes for nuclear staining.

Further, these cell lines were washed twice with an observation buffer.Then, an observation buffer containing rapamycin, or rapamycin and FK506at certain concentrations was added to each well, and incubated for 20minutes.

Note that the concentration of rapamycin added was 0.39 nM, 0.78 nM,1.56 nM, 3.13 nM, 6.25 nM, 12.50 nM, 25.00 nM, 50.00 nM, 100.00 nM,200.00 nM, 400.00 nM, or 800.00 nM. Moreover, FK506 is known as asubstance competitively inhibiting an interaction between FKBP12 andrapamycin. In this Example, FK506 was added to each well after dilutedwith a buffer containing 20 nM rapamycin in such a manner that the FK506concentration was 0.008 μM, 0.016 μM, 0.031 μM, 0.063 μM, 0.125 μM,0.250 μM, 0.500 μM, 1.000 μM, 2.000 μM, 4.000 μM, 8.000 μM, or 16.000μM.

After each drug was added and incubated, 4% Paraformaldehyde PhosphateBuffer Solution (manufactured by Wako Pure Chemical Industries, Ltd.)was added to each well, followed by incubation at room temperature for15 minutes. Thereby, these cell lines were fixed. Then, these cell lineswere washed three times with an observation buffer. Subsequently, animage of three fields of view was obtained for each well using afluorescence microscope. Thereafter, each fluorescence image wasanalyzed using iCY (see de Chaumont F et al., Nature Methods, June 28,vol. 9, no. 7, pp. 690 to 696), and a total luminance (totalfluorescence intensity) of fluorescent foci (assemblies) per cell wascalculated to analyze a correlation with the concentration of the drugsadded. FIG. 21 shows the result obtained by adding only rapamycin, andFIG. 22 shows the result obtained by adding rapamycin and FK506.

Note that, in FIGS. 21 and 22, the X axis represents the concentrationof the drug added to the cell line; the Y axis represents the totalluminance (total fluorescence intensity) of fluorescent foci(assemblies) per cell; and dots represent measurement values. Moreover,a fitting curve drawn through the dots shows the analysis result usingIgor® (manufactured by WaveMetrics, Inc.).

As apparent from the result shown in FIG. 21, it was revealed that whenthe compound rapamycin inducing an interaction between mTOR(FRB domain)and a FKBP12 protein was added to the cells stably expressing mTOR(FRBdomain)-AG and p62(PB1)-FKBP12, the fluorescence intensity offluorescent foci was increased in a manner dependent on theconcentration of rapamycin added. It was demonstrated that assembliesbetween mTOR(FRB domain)-AG and p62(PB1)-FKBP12 were formed in a mannerdependent on the concentration. Further, the EC50 of rapamycin for theprotein-protein interaction between mTOR(FRB domain) and FKBP12 was 3.36nM by the calculation according to f(x)=max+(min−max)/(1+(x/EC50)^hill)based on the fitting curve shown in FIG. 21.

On the other hand, as apparent from the result shown in FIG. 22, it wasrevealed that when FK506 was added in the presence of rapamycin, thefluorescence intensity of fluorescent foci was decreased in a mannerdependent on the concentration of FK506 added. It was demonstrated thatthe assembly formation between mTOR(FRB domain)-AG and p62(PB1)-FKBP12was inhibited in a manner dependent on the concentration. Further, theIC50 of FK506 for the interaction between rapamycin and FKBP12 andeventually for the protein-protein interaction between mTOR(FRB domain)and FKBP12 was 0.68 μM by the calculation according tof(x)=min+(max−min)/(1+(x/IC50)^hill) based on the fitting curve shown inFIG. 22.

Example 12 Detection 8 of Protein-Protein Interaction

For the same purposes as those in Examples 10 and 11, whether or notfusion proteins constituting assemblies (fluorescent foci) weredispersed by an inhibitor specific to a protein-protein interaction inthe method of the present invention, and whether or not the fluorescenceintensity of fluorescent foci was changed in a manner dependent on theinhibitor concentration by changing the inhibitor concentration weretested by a method described below.

Note that the detection target in this test was an interaction between ap53 protein and an MDM2 protein, and that Nutlin-3 known as an inhibitoragainst the interaction was used in Example 12 (see Vassilev L T et al.,Science, Feb. 6, 2004, vol. 303, no. 5659, pp. 844 to 848).

(Preparation of Plasmid DNAs)

In preparing pp62(PB1)-p53, first, a DNA encoding a portion of p53(region having the 1st to 70th amino acids of the p53 protein, theregion had the amino acid sequence of SEQ ID NO: 26) (the DNA had thebase sequence of SEQ ID NO: 25) was amplified from a cDNA library ofU2OS cells by PCR using the following primer set:

p53 forward primer; (SEQ ID NO: 97)5′-AAGGATCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCG-3′,  andp53 reverse primer48; (SEQ ID NO: 98)5′-TTGCGGCCGCTTAAGCAGCCTCTGGCATTCTGGGAGCTTCATC-3′

Then, the amplification product thus obtained was cleaved with BamHI andNotI, and inserted into pp62(PB1)-MCLinker having been treated with thesame restriction enzymes. Thus, pp62(PB1)-p53 was prepared.

Moreover, in preparing phAG-MDM2, first, a DNA encoding a portion ofMDM2 (region having the 7th to 125th amino acids of the MDM2 protein,the region had the amino acid sequence of SEQ ID NO: 28) (the DNA hadthe base sequence of SEQ ID NO: 27) was amplified from the cDNA libraryof U2OS cells by PCR using the following primer set:

MDM2 forward primer; (SEQ ID NO: 99)5′-AAGGATCCATGTGCAATACCAACATGTCTGTACCTACTGATGGTG C-3′, andMDM2 reverse primer; (SEQ ID NO: 100)5′-TTCTCGAGTTAACCTGAGTCCGATGATTCCTGCTGATTG-3′.

Then, the amplification product thus obtained was cleaved with BamHI andXhoI, and inserted into phAG-MCLinker having been treated with the samerestriction enzymes. Thus, phAG-MDM2 was prepared.

(Transfection into Cells, and Observation and Analysis of TransfectedCells)

pp62(PB1)-p53 and phAG-MDM2 were mixed in equal amounts and introducedinto HeLaS3 cells by the same method as that described in Example 1. Theculture solution was discarded 24 hours after the introduction, and 1.5ml of an observation buffer containing 0.19 μM Nutlin-3 was added to theresultant. Fluorescence images were captured 15 minutes thereafter.Subsequently, the observation buffer was discarded, and 1.5 ml of anobservation buffer containing 0.77 μM Nutlin-3 was added to theresultant. Fluorescence images were again captured 15 minutesthereafter. The same procedure was carried out using 4.8 μM and 12 μMNutlin-3, as well. FIG. 23 shows the obtained result. Moreover, a totalluminance of fluorescent foci (assemblies) in a fluorescence image ofcells to which Nutlin-3 was added at predetermined concentrations wascalculated to create a graph for illustrating a correlation with theNutlin-3 concentration. FIG. 24 shows the obtained result. Note that, inFIG. 24, the X axis represents the concentration of Nutlin-3 added tothe cells, and the Y axis represents the total luminance (totalfluorescence intensity) of fluorescent foci per fluorescence image (onefield of view).

As apparent from the result shown in FIG. 23, when p62(PB1)-p53 andAG-MDM2 were expressed in the cells, fluorescent foci were detected.Moreover, as a result of the stepwise increase in the concentration ofthe inhibitor (Nutlin-3) added against the interaction between p53 andMDM2, fluorescent foci (assemblies) were observed to be graduallyextinguished within the same field of view, confirming that the fusionproteins constituting the assemblies were being dispersed. Further, asapparent from the result shown in FIG. 24, the fluorescence luminance ofthe fluorescent foci in the field of view was decreased in a mannerdependent on the inhibitor concentration.

Example 13 Detection 9 of Protein-Protein Interaction

As in Example 11, whether or not the method of the present invention wasutilizable in determining the IC50 of an inhibitor specific to aprotein-protein interaction was evaluated by a method described below.

(Preparation of Plasmid DNA)

First, using phAG-MDM2 described in Example 12 and pFucci-S/G2/MGreen-Hyg (manufactured by limited company Amalgaam Co., Ltd.),phAG-MDM2_Hyg was prepared according to a conventional method, so thatthe drug resistance gene was converted from a G418 resistance gene to ahygromycin B resistance gene.

(Preparation of Stably-Expressing Cell Line)

Next, the phAG-MDM2_Hyg and pp62(PB1)-p53 described in Example 12 wereintroduced into a CHO-K1 cell line. Note that CHO-K1 cells were culturedin NUTRIENT MIXTURE F-12 HAM (manufactured by SIGMA ALDRICH CO.)containing 10% FBS (manufactured by Equitech-Bio Inc.).

Then, 24 hours after the plasmid DNAs were introduced in the CHO-K1 cellline, the medium was replaced with one containing 100 μg/ml of G418(manufactured by Wako Pure Chemical Industries, Ltd.) and 200 μg/ml ofhygromycin (manufactured by Nacalai Tesque, Inc.). Further, cellssurvived after culturing for one week with this medium were monoclonedby limiting dilution.

(Observation and Analysis of Transfected Cells)

After the nuclear staining on the monocloned cell line by the samemethod as that described in Example 11, an observation buffer containingNutlin-3 (manufactured by CALBIOCHEM) at certain concentrations wasadded to each well, and incubated for 20 minutes. Note that Nutlin-3 wasprepared and added in such a manner that the final concentration was 0.2μM, 0.3 μM, 0.6 μM, 0.9 μM, 1.6 μM, 2.6 μM, 4.3 μM, 7.2 μM, 12.0 μM, or20.0 μM.

Subsequently, the cell line was fixed by the same method as thatdescribed in Example 11. An image of each well was obtained with afluorescence microscope, and each fluorescence image was analyzed usingiCY. A total luminance of fluorescent foci per cell was calculated toanalyze a correlation with the concentration of Nutlin-3 added. FIG. 25shows the obtained result. Note that, in FIG. 25, the X axis representsthe concentration of the drug added to the cell line; the Y axisrepresents the total luminance (total fluorescence intensity) offluorescent foci (assemblies) per cell; and dots represent measurementvalues. Moreover, a fitting curve drawn through the dots shows theanalysis result using Igor®.

Although unillustrated, in CHO-K1 cells also stably expressingp62(PB1)-p53 and AG-MDM2, fluorescent foci (assemblies) attributable toa protein-protein interaction between p53 and MDM2 were observed as inExample 12. Moreover, as shown in FIG. 25, the fluorescence luminance ofthese assemblies was decreased in a manner dependent on theconcentration of the inhibitor Nutlin-3. Further, the IC50 of Nutlin-3for the protein-protein interaction between p53 and MDM2 was 8.9 μM bythe calculation according to f(x)=min+(max−min)/(1+(x/IC50)^hill) basedon the fitting curve shown in FIG. 25.

The results described in Examples 10 to 13 above verified that thefluorescence luminance of the fluorescent focus according to the presentinvention reflected the strength of the protein-protein interaction,making quantification of the protein-protein interaction possible.Furthermore, it was also revealed that the assembly formation wasreversible. It was demonstrated that the quantification was practicableby using fixed cells (see FIG. 20), and also using a live imagingtechnique with living cells (see FIG. 24). In addition, it was revealedas described in Examples 11 and 13 that the present invention enableddetection of a promoting reaction and an inhibiting reaction bothdependent on the concentration of a drug for a protein-proteininteraction, and further enabled calculations of the EC50 and the IC50of the drug. Thus, it was demonstrated that the method for detecting aprotein-protein interaction of the present invention was applicable toevaluation of and screening for a substance modulating a protein-proteininteraction.

Example 14 Detection 10 of Protein-Protein Interaction

It has been known that p50 and p65 form a heterodimer, constitutingNFκB. Further, NF KB functions in the nucleus as a transcription factorplaying a role in modulating inflammatory cytokine expression. However,it has been known that the interaction with IκBα retains NFκB in thecytoplasm, suppressing the transcription function (see Marc D. Jacobs etal., Cell, Dec. 11, 1998, vol. 95, pp. 749 to 758). Thus, anoverexpression of p50 and p65 disturbs the stoichiometric balance withendogenous IκBα, so that the heterodimer is localized mainly in thenucleus. On the other hand, if IκBα is overexpressed, the heterotrimerfurther including IκBα is retained in the cytoplasm.

For this reason, in this Example, whether or not the method of thepresent invention enabled detection of a change in intracellularlocalization of a complex containing p50 and p65 in accordance with thepresence or absence of IκBα was tested by a method described below.

First, each of pp62(PB1)-p50 and phAG-p65 was prepared by the samemethod as that described in Example 2. DNAs (SEQ ID NOs: 155 and 157,respectively) encoding regions having the amino acid sequences of SEQ IDNOs: 156 and 158 had been inserted in the pp62(PB1)-p50 and thephAG-p65, respectively.

Meanwhile, pIκBα was prepared by the same method as that described inExample 1 using a DNA sequence encoding the amino acid sequencespecified under Genbank ACCESSION No: NP_065390.1.

Then, HeLaS3 cells were seeded onto 4 wells in an 8-well chamber(manufactured by Nunc A/S). On the next day, the plasmid DNAs wereintroduced into these cells. In the transfection, 100 ng of each ofpp62(PB1)-p50 and phAG-p65 was added to OptiMEM (manufactured by LifeTechnologies Corporation), and pIκBα was further added thereto indifferent amounts for the use. Note that the amounts of the pIκBα addedwere 0 ng (not added) or 100 ng. Further, to make total amounts of theplasmid DNAs added all equal, 300 ng or 0 ng of pmKeima-Red-S1(manufactured by limited company Amalgaam Co., Ltd.) was added. Then,1.5 μl of PolyFect® Transfection Reagent was added to each OptiMEM andstirred. Furthermore, after mixed with 200 μl of the culture solution,the resultant was added to the HeLaS3 cells. Subsequently, 22 hoursafter this transfection, the cells were fixed with 4% ParaformaldehydePhosphate Buffer Solution (manufactured by Wako Pure ChemicalIndustries, Ltd.) at room temperature for 15 minutes. After the cellmembranes were solubilized with 0.2% TritonX-100/PBS for 5 minutes,immunostaining was carried out using an anti-IκBα antibody (manufacturedby Cell Signaling Technology, Inc.). Furthermore, the nuclei werestained using Hoechst 33342. Then, the immunostained cells were observedby the same method as that described in Example 1. FIG. 26 shows theobtained result. Note that, in the figure, “merging” shows the result ofmerging images of AG-derived fluorescence (two panels on the left in thefigure), images showing the result of the immunostaining with theanti-IκBα antibody (two panels in the middles of the figure), and imagesshowing the result of the nuclear staining with Hoechst 33342.

As apparent from the result shown in FIG. 26, it was confirmed that, inthe cells into which IκBα was not introduced, heterodimers formed fromp50 and p65 were formed in the nuclei. On the other hand, when IκBα wasintroduced, IκBα-derived signals were detected (see the lower panel inthe middle of the figure) at the same locations as the fluorescent focidetected in the image of the AG-derived fluorescence (see the lower leftpanel in the figure), confirming that the complex containing p50 and p65included IκBα. Further, the method of the present invention alsoconfirmed that in the presence of IκBα, the localization of the complexcontaining p50 and p65 was changed from the inside of the nuclei to theinside of the cytoplasms.

Example 15 Detection 11 of Protein-Protein Interaction

In this Example, utilizing the complex containing p50 and p65 detectedalso in Example 14, whether or not the method of the present inventionenabled quantitative detection of a change in intracellular localizationof the complex in accordance with the quantitative balance with IκBα wastested by a method described below.

First, HeLaS3 cells were seeded onto 4 wells in an 8-well chamber(manufactured by Nunc A/S). Then, on the next day, pp62(PB1)-p50,phAG-p65, and pIκBα, which were described in Example 13, were introducedinto these cells. In the transfection, 100 ng of each of pp62(PB1)-p50and phAG-p65 added to OptiMEM (manufactured by Life TechnologiesCorporation), and the pIκBα was further added thereto in differentamounts for the use. Note that the amounts of the pIκBα added were (0)not added, (1) 33 ng, (2) 100 ng, and (3) 300 ng. Further, to make totalamounts of the plasmid DNAs added all equal, pmKeima-Red-S1(manufactured by limited company Amalgaam Co., Ltd.) was added inamounts of: 300 ng in the case of (0); 267 ng, (1); 200 ng, (2); and 0ng, (3). Then, 1.5 μl of PolyFect® Transfection Reagent was added toeach OptiMEM and stirred. Furthermore, after mixed with 200 μl of theculture solution, the resultant was added to the HeLaS3 cells, andobserved 22 hours thereafter by the same method as that described inExample 1.

After 150 or more cells in which the plasmid DNAs were introduced underthe (0) to (3) conditions were photographed, the cells were classifiedinto three groups (A) to (C) according to the fluorescent focuslocalization. Specifically, cells are classified as (A) if fluorescentfoci were detected only in the cytoplasms; (B), if detected in thecytoplasms and the nuclei; and (C), if detected only in the nuclei.Then, a percentage of the cell count in each group was calculated, and agraph was created. FIG. 27 shows the obtained result.

As apparent from the result shown in FIG. 27, the percentage offluorescent foci detected in the cytoplasms was increased in a mannerdependent on the IκBα amount. Thus, the present invention can provide amethod for grouping cells according to fluorescent focus localization,and comparing the number of cells in each group. Further, it wasrevealed as described in Example 14 also that by utilizing localizationof an interaction between direct detection targets, a first protein anda second protein (for example, p50 and p65), the method of the presentinvention enabled quantification of an influence of a third protein (forexample, IκBα) on the interaction as well as the amount of the thirdprotein expressed.

Example 16 Detection 12 of Protein-Protein Interaction

It has been known that, in the nucleus of a cell, p21 recognizes andinteracts with a complex composed of CDK4 and Cyclin D1 (see LaBaer J etal., Genes Dev., Apr. 1, 1997, vol. 11, no. 7, pp. 847 to 862).Moreover, it has also been revealed that such heterotrimer formationinhibits cell-cycle progression (transition from a G1 phase to an Sphase) otherwise promoted by a complex composed of CDK4 and Cyclin D1.

For this reason, in this Example, as in Examples 14 and 15, whether ornot the present invention enabled detection of formation of a complexcomposed of three types of different proteins was tested by a methoddescribed below.

(Preparation of Plasmid DNA)

pp62(PB1)-CDK4, phAG-p21, and pCyclin D1 were prepared by the samemethod as that described in Example 1 on the basis of DNA sequencesencoding the amino acid sequences specified under Genbank ACCESSION Nos:NP_000066.1, NP_000380.1, and NP_444284.1, respectively.

(Transfection into Cultured Cells, Cell Immunostaining, and Observationof Cells)

HeLaS3 cells were used as cultured cells into which the plasmid DNAswere introduced. Moreover, the HeLaS3 cells were cultured by the samemethod as that described in Example 1. Further, in the transfection, theHeLaS3 cells were seeded onto 2 wells of an 8-well chamber (manufacturedby Nunc A/S). On the next day, by the same method as that described inExample 1, 130 ng of each plasmid DNA in the following combinations wasintroduced into the HeLaS3 cells using 1 μl of Transfection Reagent:

a combination of pp62(PB1)-CDK4 and phAG-p21 with pCyclin D1; and

a combination of pp62(PB1)-CDK4 and phAG-p21 with phmKGC-MN(manufactured by limited company Amalgaam Co., Ltd.) (note that thephmKGC-MN was added to makes total amounts of the plasmids all equal).

Then, 24 hours after the transfection, the cells were fixed with 4%Paraformaldehyde Phosphate Buffer Solution (manufactured by Wako PureChemical Industries, Ltd.) at room temperature for 15 minutes. After thecell membranes were solubilized with 0.2% TritonX-100/PBS for 5 minutes,immunostaining was carried out using 4 μg/ml of an anti-Cyclin D1antibody (manufactured by MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.).The immunostained cells were observed by the same method as thatdescribed in Example 1. FIG. 28 shows the obtained result.

As apparent from the result shown in FIG. 28, no clear assembly(fluorescent focus) was observed in many cells only by expressingp62(PB1)-CDK4 and AG-p21. Nevertheless, when Cyclin D1 was forciblyexpressed together with p62(PB1)-CDK4 and AG-p21, this made possiblestoichiometrically uniform expression of the elements necessary for theheterotrimer formation, and clear fluorescent foci were observed inalmost all the cells. Further, the immunostaining images of Cyclin D1confirmed that Cyclin D1 was localized in the observed fluorescent foci.

Thus, similarly to the results described in Examples 14 and 15, it wasconfirmed that the present invention enabled detection of aprotein-protein interaction in formation of a complex of a trimer or ahigher multimer.

Moreover, suppose a case where two types of proteins (p50 and p65 inExamples 14 and 15, CDK4 and p21 in Example 16) are expected to form acomplex including a “certain molecule” (IκBα in Examples 14 and 15,Cyclin D1 in Example 16) as described above. In this case, if the twotypes of proteins are expressed in cells as a “first fusion proteincomprising an association-inducing protein” and a “second fusion proteincomprising a fluorescent protein having a multimerization ability”respectively, and a protein encoded by a cDNA expression library isfurther expressed in the cells, the present invention makes it possibleto search for the “certain molecule” (constitutional factor of thecomplex) on the basis of a fluorescent focus (for example, formation orextinction a fluorescent focus, a change in fluorescent focuslocalization).

Further, as described in Examples 14 to 16, the present invention makesit possible to analyze an expressed amount of a constitutional factor(IκBα in Examples 14 and 15, Cyclin D1 in Example 16) of a complex onthe basis of a fluorescent focus, and eventually, by utilizing theexpressed amount, to analyze states of cells, such as a cell cyclecontrolled by the complex and a stress to which the complex is torespond.

Example 17 Detection 13 of Protein Interaction

As described in Examples 10 to 13, it was revealed that the fluorescenceintensity of the fluorescent focus according to the present inventionreflected a strength of a protein-protein interaction. Thus, the methodfor detecting a protein-protein interaction of the present inventionpresumably is capable of identifying an amino acid important for theinteraction on the basis of the fluorescence intensity of thefluorescent focus according to the present invention. For this reason, atest was conducted by a method described below, utilizing an amino acidmutation known to be involved in reduction or enhancement of aprotein-protein interaction.

Note that the detection target in this test was an interaction between aSec5 protein and a RalB protein. A Sec5 protein has been known tointeract with a RalB protein in a GTP-activated form (see Moskalenko Set al., Nat Cell Biol., January 2002, vol. 4, no. 1, pp. 66 to 72). Ithas been known that the interaction is reduced with an inactive mutantRalB(S28N) of RalB, but enhanced with an active mutant RalB(Q72L) ofRalB (see Shipitsin M et al., Mol Cell Biol., July 2004, vol. 24, iss.13, pp. 5746 to 5756). Further, a RalB protein, which is amembrane-anchored protein, has been revealed to be localized at the cellmembranes by palmitoylation of the C-terminus thereof.

(Preparation of Plasmid DNA)

In preparing pp62(PB1)-Sec5, first, a DNA encoding a portion of Sec5(region having the 1st to 99th amino acids of the Sec5 protein, theregion had the amino acid sequence of SEQ ID NO: 30) (the DNA had thebase sequence of SEQ ID NO: 29) was amplified from the cDNA library ofHeLaS3 cells by PCR using the following primer set:

Sec5 forward primer; (SEQ ID NO: 101)5′-CCCGGATCCATGTCTCGATCACGACAACCC-3′, and Sec5 reverse primer;(SEQ ID NO: 102) 5′-GGGAAGCTTTTATTAGCCTATTTTCTCAGGTTTGAGTA-3′.

Then, the amplification product thus obtained was cleaved with BamHI andHindIII, and inserted into pp62(PB1)-MCLinker having been treated withthe same restriction enzymes. Thus, pp62(PB1)-Sec5 was prepared. Notethat the pp62(PB1)-Sec5 encodes a fusion protein composed of p62(PB1)and a partial Sec5 protein (the fusion protein may also be referred toas “p62(PB1)-Sec5”).

Meanwhile, in preparing phAG-RalB(WT), first, a DNA encoding RalB(protein having the amino acid sequence of SEQ ID NO: 32) (the DNA hadthe base sequence of SEQ ID NO: 31) was amplified from the cDNA libraryof HeLaS3 cells by PCR using the following primer set:

RalB forward primer; (SEQ ID NO: 103)5′-CCCGGATCCATGGCTGCCAACAAGAGTAAG-3′, and RalB reverse primer;(SEQ ID NO: 104) 5′-GGGAAGCTTTTATCATAGTAAGCAACATCTTTC-3′.

Then, the amplification product thus obtained was cleaved with BamHI andHindIII, and inserted into phAG-MCLinker having been treated with thesame restriction enzymes. Thus, phAG-RalB(WT) was prepared. Note thatthe phAG-RalB(WT) encodes a fusion protein composed of an AG protein anda RalB protein (the fusion protein may also be referred to as“AG-RalB(WT)”).

Further, phAG-RalB(Q72L) was prepared using phAG-RalB(WT) as a template,and AMAP™ Multi Site-directed Mutagenesis Kit (manufactured by limitedcompany Amalgaam Co., Ltd.), and according to the attached instruction,a mutation was introduced using the following primer:

RalB (Q72L) primer; (SEQ ID NO: 105)5′-CTGGACACCGCTGGGCTAGAGGACTACGCAGCCA-3′.

Note that the amino acid sequence of a RalB(Q72L) protein is shown inSEQ ID NO: 34. Moreover, the base sequence of a DNA encoding the proteinis shown in SEQ ID NO: 33. Further, phAG-RalB(Q72L) encodes a fusionprotein composed of an AG protein and a RalB(Q72L) protein (the fusionprotein may also be referred to as “AG-RalB(Q72L)”).

In addition, phAG-RalB(S28N) was prepared using phAG-RalB(WT) as atemplate, and AMAP™ Multi Site-directed Mutagenesis Kit, and accordingto the attached instruction, a mutation was introduced using thefollowing primer:

RalB (S28N) primer ; (SEQ ID NO: 106)5′-CAGCGGAGGCGTTGGCAAGAACGCCCTGACGCTTCAGTTCA-3′.

Note that the amino acid sequence of a RalB(S28N) protein is shown inSEQ ID NO: 36. Moreover, the base sequence of a DNA encoding the proteinis shown in SEQ ID NO: 35. phAG-RalB(S28N) encodes a fusion proteincomposed of an AG protein and a RalB(S28N) protein (the fusion proteinmay also be referred to as “AG-RalB(S28N)”).

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into HeLaS3 cells by the same method asthat described in Example 1:

a combination of pp62(PB1)-Sec5 with phAG-RalB(WT);

a combination of pp62(PB1)-Sec5 with phAG-RalB(Q72L);

a combination of pp62(PB1)-Sec5 with phAG-RalB(S28N); and

a combination of pp62(PB1) with phAG-RalB(WT).

Moreover, the transfected cells were observed also by the same method asthat described in Example 1. FIG. 29 shows the obtained result. Further,images were obtained using a total internal reflection fluorescencemicroscopy system with arc lamp source (manufactured by OlympusCorporation, IX71-ARCEVA) capable of exciting only the vicinity of thecell membrane. FIG. 30 shows the obtained result.

As apparent from the result shown in FIG. 29, since the RalB proteinhaving the C-terminus palmitoylated was localized at the cell membranes,when the wildtype RalB protein (RalB(WT)) was expressed, fluorescentfoci derived from the interaction with the Sec5 protein were detected inthe vicinity of the cell membranes. On the other hand, no fluorescentfocus was detected in the cells co-expressing p62(PB1)-Sec5 andAG-RalB(S28N). It was confirmed as described above that the inactivemutant RalB(S28N) of RalB reduced the interaction with the Sec5 protein.Moreover, in the cells co-expressing p62(PB1)-Sec5 and AG-RalB(Q72L),fluorescent foci were detected, which had a higher fluorescenceintensity (luminance) than that in the cells co-expressing p62(PB1)-Sec5and AG-RalB(WT). It was also confirmed as described above that theactive mutant RalB(Q72L) of RalB enhanced the interaction with the Sec5protein.

Further, as apparent from the result shown in FIG. 30, in the cellsco-expressing p62(PB1)-Sec5 and AG-RalB(WT), assemblies were observed.The result shown in FIG. 30 was a result of using the observation systemcapable of exciting only the vicinity of the cell membrane. Thus,similarly to the result shown in FIG. 29, it was verified that themethod of the present invention enabled detection of the interactionbetween the wildtype RalB protein and the Sec5 protein in the vicinityof the cell membranes.

Furthermore, as apparent from the result shown in FIG. 30, in the cellsco-expressing p62(PB1)-Sec5 and AG-RalB(Q72L), more significant assemblyformation was observed. On the other hand, in the cells co-expressingp62(PB1)-Sec5 and AG-RalB(S28N), no assembly was observed. Thus,similarly to the result shown in FIG. 29, it was verified that themethod of the present invention enabled detection of reduction of theinteraction with the Sec5 protein by RalB(S28N), and enhancement of theinteraction by RalB(Q72L).

Example 18 Detection 14 of Protein Interaction

As in Example 17, a test was conducted by a method described below,using an amino acid mutation known to be involved in reduction of aprotein-protein interaction.

(Preparation of Plasmid DNA)

pp62(PB1)-p53_W23L was prepared by introducing a mutation intopp62(PB1)-p53 described in Example 12 by the same method as thatdescribed in Example 17 using a primer having the base sequence of SEQID NO: 159 (5′-ACATTTTCAGACCTATTGAAACTACTTCCTGAAAACAACGT-3′).

Note that the amino acid at position 23 of p53 is located at aninteraction interface site between p53 and MDM2. Further, a “W23L”mutation of p53 has been known as a mutation resulting in reduction ofthe interaction (see literature Zondlo S C, Biochemistry, Oct. 3, 2006,vol. 45, no. 39, pp. 11945 to 11957).

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into cells by the same method as thatdescribed in Example 1, and the cells were observed. FIG. 31 shows theobtained result.

A combination of pp62(PB1)-p53 with phAG-MDM2.

A combination of pp62(PB1)-p53_W23L with phAG-MDM2.

As apparent from the result shown in FIG. 31, in the cells co-expressingp62(PB1)-p53 and AG-MDM2, fluorescent foci (assembly formation) weresignificantly observed (the left panel in the figure). On the otherhand, in the cells co-expressing p62(PB1)-p53_W23L and AG-MDM2, noassembly was observed (the right panel in the figure).

Thus, as described in Examples 17 and 18, it was revealed that themethod for detecting a protein-protein interaction of the presentinvention was also capable of identifying the amino acid important forthe interaction on the basis of the fluorescence intensity of thefluorescent focus according to the present invention. Particularly, themethod of the present invention is capable of very easily specifying anamino acid involved in a protein-protein interaction by introducing amutation into an interface of the interaction and detecting the presenceor absence of the interaction. In other words, combining a method forintroducing a mutation into a protein such as alanine scanning with themethod of the present invention makes it possible to search for a site(hot spot) important for a protein interaction very easily.

Example 19 Detection 15 of Protein-Protein Interaction

As described above, it was demonstrated that the method for detecting aprotein-protein interaction of the present invention enabledquantitative measurement of when a protein-protein interaction tookplace and ended in real time. Hence, a test was conducted regardingwhether or not the use of the method of the present invention enableddetection of how an endogenous signal transduction in cells changed overtime, according to a protein-protein interaction that took place inresponse to the signal.

Specifically, the protein-protein interaction as the detection target inthis test was an interaction between calmodulin and a partial sequence(M13 peptide) of myosin light chain kinase 2. It has been revealed thatthe interaction takes place in response to a transient increase inintracellular calcium ion concentration (second messenger) that occurswhen a G protein-coupled receptor (GPCR) receives a ligand (see MiyawakiA et al., Nature, Aug. 28, 1997, vol. 388, no. 6645, pp. 882 to 887).Hence, whether or not it was possible to detect a change inintracellular calcium ion concentration over time according to theinteraction was tested by a method described below. FIG. 32 shows theobtained result.

(Preparation of Plasmid DNAs)

In preparing pCalmodulin-hAG, first, a DNA encoding calmodulin (proteinhaving the amino acid sequence of SEQ ID NO: 38) (the DNA had the basesequence of SEQ ID NO: 37) was amplified from the cDNA library of HeLaS3cells by PCR using the following primer set:

calmodulin forward primer; (SEQ ID NO: 107)5′-TTGGATCCGCCACCATGGACCAACTGACAGAAGAGCAGATTGC-3′, andcalmodulin reverse primer; (SEQ ID NO: 108)5′-AAGAATTCCCCTTTGCTGTCATCATTTGTACAAACTCTTC-3′

Then, the amplification product thus obtained was cleaved with BamHI andEcoRI, and inserted into phAG-MNLinker having been treated with the samerestriction enzymes. Thus, pCalmodulin-hAG was prepared. Note that thepCalmodulin-hAG encodes a fusion protein composed of a calmodulinprotein and an AG protein (the fusion protein may also be referred to as“Calmodulin-AG”).

Meanwhile, in preparing pM13peptide-p62(PB1), first, a DNA encoding aportion of myosin light chain kinase 2 (region having the 566th to 591stamino acids of the myosin light chain kinase 2 protein, the region hadthe amino acid sequence of SEQ ID NO: 40) (the DNA had the base sequenceof SEQ ID NO: 39) was amplified from the cDNA library of HeLaS3 cells byPCR using the following primer set:

M13 peptide forward primer; (SEQ ID NO: 109)5′-TTGGATCCGCCACCATGAAGAGGCGCTGGAAGAAAAACTTCATTG C-3′, andM13 peptide reverse primer; (SEQ ID NO: 110)5′-CCGAATTCCCCAGTGCCCCGGAGCTGGAGATCTTCTTG-3′.

Then, the amplification product thus obtained was cleaved with BamHI andEcoRI, and inserted into pp62(PB1)-MNLinker having been treated with thesame restriction enzymes. Thus, pM13peptide-p62(PB1) was prepared. Notethat the pM13peptide-p62(PB1) encodes a fusion protein composed of anM13 peptide and p62(PB1) (the fusion protein may also be referred to as“M13peptide-p62(PB1)”).

(Transfection into Cultured Cells, and Observation of Transfected Cells)

pCalmodulin-hAG and pM13peptide-p62(PB1) were mixed in equal amounts andintroduced into HeLaS3 cells by the same method as that described inExample 1. Then, 200 μM histamine (manufactured by Wako Pure ChemicalIndustries, Ltd.) was added thereto, and fluorescence images werecaptured over time. Note that it has been revealed that histaminefunctions as a ligand of an H1 receptor, one of GPCRs, which isexpressed also in HeLaS3 cells.

As apparent from the result shown in FIG. 32, before the ligand(histamine) was added, Calmodulin-AG was present in a dispersed mannerin the cells. Meanwhile, 90 seconds after the ligand was added,fluorescent focus formation was detected, confirming the assemblyformation with M13peptide-p62(PB1). Nevertheless, 490 seconds after thecytoplasm calcium ion concentration was decreased, the fluorescent foci(assemblies) were extinguished.

Thus, it was revealed that the use of the method for detecting aprotein-protein interaction of the present invention enabled real-timemeasurement of the calcium ion concentration transiently increased bythe signal transduction from the H1 receptor.

Moreover, the result of this Example 19 also demonstrated that thepresent invention enabled detection of the transient protein-proteininteraction, further that the present invention was applicable todetection of and screening for: an endogenous factor such as a secondmessenger causing a protein-protein interaction; a signal transductionto which the second messenger or the like contributed; and a stimulusfrom the outside such as an extracellular ligand eliciting the signaltransduction.

Example 20 Detection 16 of Protein-Protein Interaction

In conventional methods for detecting a protein-protein interactionrepresented by WO2000/017221 A and WO2006/099486 A, one of proteinsconstituting a complex formed by a protein-protein interaction isforcibly (artificially) confined in a particular region in a cell.Accordingly, the detection was impossible in an intracellularenvironment unique to the interaction. In contrast, in the method fordetecting a protein-protein interaction of the present invention,fluorescent foci (assemblies) are autonomously formed only when aninteraction takes place. Hence, it is expected that the problems in theconventional method can be solved. For this reason, whether or not thepresent invention enabled detection of an interaction in any region in acell was tested by a method described below.

(Preparation of Plasmid DNA)

In preparing pmTOR(FRB domain)-AGNLS, first, an AGNLS gene was amplifiedfrom pNP-AG (manufactured by limited company Amalgaam Co., Ltd.) by PCRusing the following primer set:

AGNLS forward primer; (SEQ ID NO: 111)5′-AAACCGGTATGGTGAGTGTGATTAAACCAGAG-3′, and AGNLS reverse primer;(SEQ ID NO: 112) 5′-AATCTAGATTATTTATCCTTTTCCTTTTTACTCTTCTTCTTAGCTACTTC-3′.

Then, the amplification product thus obtained was cleaved with AgeI andXbaI, and inserted into pmTOR(FRB domain)-hAG having been treated withthe same restriction enzymes to cut out a hAG region therefrom. Thus,pmTOR(FRB domain)-AGNLS was prepared. Note that the pmTOR(FRBdomain)-AGNLS encodes a fusion protein composed of mTOR(FRB domain), anAG protein, and a nuclear localization signal (NLS) (the fusion proteinmay also be referred to as “mTOR(FRB domain)-AGNLS”). Moreover, mTOR(FRBdomain)-AGNLS is to be localized in the nucleus of a cell because thenuclear localization signal is fused to the C-terminus of the mTOR(FRBdomain)-AG.

Meanwhile, in preparing pp62(PB1)-HRas, first, a DNA encoding an HRasprotein was amplified from the cDNA library of HeLaS3 cells by PCR usingthe following primer set. The amplification product thus obtained wascleaved with EcoRI and XhoI.

HRas forward primer; (SEQ ID NO: 113)5′-AAGAATTCGATGACGGAATATAAGCTGGTGGTGGTGGGCGCCGTCG GTGTGGGCAAGAGTGC-3′,and HRas reverse primer; (SEQ ID NO: 114)5′-TTCTCGAGACCTCCGGAGACGTTCAGCTTCCGCAGCTTGTGCTGCC GGATCTCACGCACCAAC-3′.

Further, a prenylated sequence derived from a KRas protein was amplifiedby PCR using the following primer set. The amplification product thusobtained was cleaved with XhoI and NotI.

KRas forward primer; (SEQ ID NO: 115)5′-AACTCGAGAAGATGAGCAAAGATGGTAAAAAGAAGAAAAAGAAGTC AAAGACAAAGTGTG-3′, andKRas reverse primer; (SEQ ID NO: 116)5′-TTGCGGCCGCTTACATAATTACACACTTTGTCTTTGACTTCTTTTT CTTCTTTTTACCAT-3′.

Then, the two DNA fragments prepared in this manner were inserted intopp62(PB1)-MCLinker having been treated with EcoRI and NotI. Thus,pp62(PB1)-HRas(WT) was prepared.

Furthermore, pp62(PB1)-HRas encoding a fusion protein composed of aconstitutively-active mutant HRas and p62(PB1) was prepared byintroducing a mutation using pp62(PB1)-HRas(WT) as a template, and AMAP™Multi Site-directed Mutagenesis Kit (manufactured by limited companyAmalgaam Co., Ltd.) according to the attached instruction, with thefollowing primer:

HRas mutant primer; (SEQ ID NO: 117)5′-GCTGGTGGTGGTGGGCGCCGTCGGTGTGGGCAAGAGTGCGC-3′.

Note that the pp62(PB1)-HRas encodes a protein obtained by fusingp62(PB1) with a DNA encoding HRas having the C-terminus to which theKRas protein-derived prenylated sequence is added (the protein had theamino acid sequence of SEQ ID NO: 42) (the DNA had the base sequence ofSEQ ID NO: 41). Moreover, since having the prenylated sequence, thisfusion protein is subjected to post-translation lipid modification incells, and localized at the cell membranes.

In preparing phAG-cRaf, first, a DNA encoding a portion of cRaf (regionhaving the 51st to 131st amino acids of the cRaf protein, the region hadthe amino acid sequence of SEQ ID NO: 44) (the DNA had the base sequenceof SEQ ID NO: 43) was amplified by PCR using the cDNA library of HeLaS3cells as a template and the following primer set:

cRaf forward primer; (SEQ ID NO: 118)5′-AAGGTACCCCTTCTAAGACAAGCAACACTATCCGTGTTTTCTTGCC GAACAAGCAAAGAA-3′, andcRaf reverse primer 71; (SEQ ID NO: 119)5′-TTAAGCTTTTACAGGAAATCTACTTGAAGTTCTTCTCCAATCAAAG ACGCAG-3′.

Then, the amplification product thus obtained was cleaved with KpnI andHindIII, and inserted into phAG-MCLinker having been treated with thesame restriction enzymes. Thus, phAG-cRaf was prepared. Note that thephAG-cRaf is a fusion protein composed of an AG protein and a portion ofa cRaf protein (the fusion protein may also be referred to as“AG-cRaf”). Moreover, the portion of a cRaf protein has been known tointeract with an HRas protein (see Mochizuki N et al., Nature, Jun. 28,2001, vol. 411, no. 6841, pp. 1065 to 1068).

Further, in preparing pSmac-p62(PB1), first, a DNA encoding a fusionprotein composed of a portion of Smac (region having the 1st to 10thamino acids of the Smac protein, the region had the amino acid sequenceof SEQ ID NO: 46) and p62(PB1) was amplified from pp62(PB1)-MNL by PCRusing the following primer set:

Smac forward primer; (SEQ ID NO: 120)5′-AGGATCCGCCACCATGGCCGTGCCCATCGCCCAGAAATCAGAGAAT TCGG-3′, andp62 (PB1) reverse primer 2; (SEQ ID NO: 64)5′-ACCTCTAGATTATTTCTCTTTAATGTAGATTCGGAAGATG-3′

Then, the amplification product thus obtained was cleaved with BamHI andXbaI, and inserted into pp62(PB1)-MNL having been treated with the samerestriction enzymes to cut out the linker and p62(PB1) therefrom. Thus,pSmac-p62(PB1) was prepared. Note that the pSmac-p62(PB1) encodes afusion protein composed of a portion of Smac and p62(PB1) (the fusionprotein may also be referred to as “Smac-p62(PB1)”).

In addition, in preparing pXIAP-hAG, first, a DNA encoding a portion ofXIAP (region having the 243rd to 356th amino acids of the XIAP protein,the region had the amino acid sequence of SEQ ID NO: 48) (the DNA hadthe base sequence of SEQ ID NO: 47) was amplified from the cDNA libraryof HeLaS3 cells by PCR using the following primer set:

XIAP forward primer; (SEQ ID NO: 121)5′-TTGGATCCGCCACCATGGCTGTGAGTTCTGATAGGAATTTCCCAAA TTC-3′, andXIAP reverse primer; (SEQ ID NO: 122)5′-TTGAATTCTCAGTAGTTCTTACCAGACACTCCTCAAGTGAATGA G-3′.

Then, the amplification product thus obtained was cleaved with BamHI andEcoRI, and inserted into phAG-MNLinker having been treated with the samerestriction enzymes. Thus, pXIAP-hAG was prepared. Note that thepXIAP-hAG encodes a fusion protein composed of a portion of XIAP and anAG protein (the fusion protein may also be referred to as “XIAP-AG”).Moreover, it has been known that the portion of Smac and the portion ofXIAP interact with each other in the cytoplasm (see Liu Z et al.,Nature, Dec. 21-28, 2000, vol. 408, no. 6815, pp. 1004 to 1008).

Further, in preparing pp62(PB1)-BclX(L), first, a DNA encoding a portionof BclX(L) (region having the 1st to 209th amino acids of the BclX(L)protein, the region had the amino acid sequence of SEQ ID NO: 50) (theDNA had the base sequence of SEQ ID NO: 49) was amplified from the cDNAlibrary of HeLaS3 cells by PCR using the following primer set:

BclX (L) forward primer; (SEQ ID NO: 123)5′-TTCTCGAGGATGTCTCAGAGCAACCGGGAGCTGGTGGTTGAC-3′,  andBclX (L) reverse primer; (SEQ ID NO: 124)5′-CTAAGCGGCCGCTTAGCGTTCCTGGCCCTTTCGGCTCTCGGCTG-3′.

Then, the amplification product thus obtained was cleaved with XhoI andNotI, and inserted into pp62(PB1)-MCLinker having been treated with thesame restriction enzymes. Thus, pp62(PB1)-BclX(L) was prepared. Notethat the pp62(PB1)-BclX(L) encodes a fusion protein composed of p62(PB1)and a portion of BclX(L) (the fusion protein may also be referred to as“p62(PB1)-BclX(L)”).

In addition, in preparing phAG-BAD, first, a DNA encoding a portion ofBAD (region having the 103rd to 127th amino acids of the BAD protein,the region had the amino acid sequence of SEQ ID NO: 52) (the DNA hadthe base sequence of SEQ ID NO: 51) was amplified by PCR using thefollowing primer set:

BAD forward primer 1; (SEQ ID NO: 125)5′-GCAGCACAGCGCTATGGCCGCGAGCTCCGGAGGATGAGTGACGAGT TTGT-3′,BAD forward primer 2; (SEQ ID NO: 126)5′-TTGGATCCAACCTCTGGGCAGCACAGCGCTATGGCCGCGAGCTCCG GAGG-3′, andBAD reverse primer; (SEQ ID NO: 127)5′-TTGAATTCTTACTTCTTAAAGGAGTCCACAAACTCGTCACTCATCC TCCG-3′.

Then, the amplification product thus obtained was cleaved with BamHI andEcoRI, and inserted into phAG-MCLinker having been treated with the samerestriction enzymes. Thus, phAG-BAD was prepared. Note that the phAG-BADencodes a fusion protein composed of an AG protein and a portion of BAD(the fusion protein may also be referred to as “AG-BAD”). Moreover, ithas been known that the portion of BclX(L) and the portion of BADinteract with each other in the cytoplasm (see Sattler M et al.,Science, Feb. 14, 1997, vol. 275, no. 5302, pp. 983 to 986).

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into HeLaS3 cells by the same method asthat described in Example 1:

a combination of pFKBP12-p62(PB1) with pmTOR(FRB domain)-AGNLS;

a combination of pp62(PB1)-HRas with phAG-cRaf;

a combination of pSmac-p62(PB1) with pXIAP-hAG; and

a combination of pp62(PB1)-BclX(L) with phAG-BAD.

Moreover, the transfected cells were observed also by the same method asthat described in Example 1. Nevertheless, regarding the cells in whichthe pFKBP12-p62(PB1) and the pmTOR(FRB domain)-AGNLS were introduced,fluorescence images and phase contrast images were captured 300 secondsafter 100 nM rapamycin was added. FIGS. 33 to 36 show the obtainedresults.

As apparent from the result shown in FIG. 33, when mTOR(FRBdomain)-AGNLS and FKBP12-p62(PB1) were expressed in the cells, mTOR(FRBdomain)-AGNLS was localized in a dispersed state because the mTOR(FRBdomain)-AGNLS had the nuclear localization signal sequence at theC-terminus thereof (see the left panel in FIG. 33). Moreover, by theaddition of rapamycin, fluorescent foci formed by an association betweenFKBP12-p62(PB1) and mTOR(FRB domain)-AGNLS were detected only in thenuclei (see two panels on the right in FIG. 33).

Further, as apparent from the result shown in FIG. 34, whenp62(PB1)-HRas having the prenylated sequence at the C-terminus thereofand AG-cRaf were expressed in the cells, by the prenylated sequence,fluorescent foci formed by an association between the p62(PB1)-HRas andthe AG-cRaf were detected from the cell membranes.

Furthermore, as apparent from the result shown in FIG. 35, whenSmac-p62(PB1) and XIAP-AG were expressed in the cells, fluorescent fociformed by an association between Smac-p62(PB1) and XIAP-AG were alsodetected in the cytoplasms, reflecting the interaction between the Smacprotein and the XIAP protein, which had been known to take place in thecytoplasm.

Additionally, as apparent from the result shown in FIG. 36, whenp62(PB1)-BclX(L) and AG-BAD were expressed in the cells, fluorescentfoci formed by an association between p62(PB1)-BclX(L) and AG-BAD weredetected also in the cytoplasms, reflecting the interaction between theBclX(L) protein and the BAD protein, which had been known to take placein the cytoplasm.

Example 21 Detection 17 of Protein-Protein Interaction

As in the case of Example 20, whether or not the use of the method ofthe present invention enabled detection of a protein-protein interactionin an intracellular environment unique to a target protein was tested bya method described below.

Note that, in this Example 21, the detection target was aprotein-protein interaction between a Rac1 protein and PBD (p21 bindingdomain). The Rac1 protein is a low-molecular-weight G protein, andguanine nucleotide exchange factors (GEFs) such as Tiam1, Trio, and VAV1convert the Rac1 protein from an inactive GDP-bound form to an activeGTP-bound form. Moreover, it has been known that an active Rac1 proteinand PBD of a Cdc42/Rac effector protein (p21-activated kinase 1: PAK1)interact with each other. Further, GEFs are localized differentlydepending on the type, and are localized inside the nucleus, at theborder (near the cell membrane), and so on, of a cell. For this reason,while a Rac1 protein is present everywhere in the cell, an activation ofthe Rac1 protein and an interaction between an active Rac1 protein andPBD take place in intracellular regions in accordance with GEFslocalized differently depending on the type (see Benard V et al., J BiolChem., May 7, 1999, vol. 274, no. 19, pp. 13198 to 13204).

(Preparation of Plasmid DNA)

In preparing phAG-Rac1, first, a DNA encoding a Rac1 protein (proteinhaving the amino acid sequence of SEQ ID NO: 54) (the DNA had the basesequence of SEQ ID NO: 53) was amplified from the cDNA library of HeLaS3cells by PCR using the following primer set:

Rac1 forward primer; (SEQ ID NO: 128)5′-GAGAATTCGATGCAGGCCATCAAGTGTGTGGTGG-3′, and Rac1 reverse primer;(SEQ ID NO: 129) 5′-GGCTCGAGTTACAACAGCAGGCATTTTCTCTTCC-3′.

Then, the amplification product thus obtained was cleaved with EcoRI andXhoI, and inserted into phAG-MNLinker having been treated with the samerestriction enzymes. Thus, phAG-Rac1 was prepared.

Meanwhile, in preparing pp62(PB1)-PBD, first, a DNA encoding PBD (regionhaving the 67th to 150th amino acids of the PAK1 protein, the region hadthe amino acid sequence of SEQ ID NO: 56) (the DNA had the base sequenceof SEQ ID NO: 55) was amplified from the cDNA library of HeLaS3 cells byPCR using the following primer set:

PBD forward primer; (SEQ ID NO: 130)5′-TTGGATCCAAGAAAGAGAAAGAGCGGCCAGAGATTTCTCTCCC-3′, andPBD reverse primer; (SEQ ID NO: 131)5′-CCGAATTCTTACGCTGACTTATCTGTAAAGCTCATGTATTTCTGG C-3′.

Then, the amplification product thus obtained was cleaved with BamHI andEcoRI, and inserted into pp62(PB1)-MCLinker having been treated with thesame restriction enzymes. Thus, pp62(PB1)-PBD was prepared.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

Each of the following combinations of the plasmid DNAs was mixed inequal amounts and introduced into U2OS cells by the same method as thatdescribed in Example 1:

a combination of phAG-Rac1 with pp62(PB1)-PBD; and

a combination of phAG-Rac1 with pp62(PB1)-MNLinker.

FIGS. 37 to 39 show the obtained results.

As apparent from the results shown in FIGS. 37 and 38, when AG-Rac1 andp62(PB1)-PBD were expressed in the cells, fluorescent foci formed by anassociation between AG-Rac1 and p62(PB1)-PBD were detected in the nuclei(FIG. 37) and at the borders of the cells (FIG. 38).

On the other hand, as apparent from the result shown in FIG. 39, whenAG-Rac1 and p62(PB1) not fused to PBD were expressed in the cells, nofluorescent focus was detected.

The above results demonstrated that it was possible to detect the sameprotein-protein interaction in multiple regions in the cell. Thus, itwas revealed that without forcibly (artificially) confining in aparticular region in a cell a protein constituting a complex formed by aprotein-protein interaction, the present invention enabled detection ofthe protein-protein interaction in a unique intracellular environment inaccordance with the localization of the protein.

Moreover, it was also demonstrated that by detecting the protein-proteininteraction, the present invention enabled detection of the active(GTP-bound form) Rac1 protein. Further, it was verified that it was alsopossible to detect the localization and activity of the intracellularenzyme GEF by detecting the conversion to the active GTP-bound form,thus revealing that the method of the present invention enableddetection of an activity of an endogenous factor according to aprotein-protein interaction.

Example 22 Detection 18 of Protein-Protein Interaction

As described in Example 21, it has been revealed that an active Rac1protein and PBD of a Cdc42/Rac effector protein (p21-activated kinase 1:PAK1) interact with each other.

Moreover, it has also been known that the localization of a Rac1 proteinis changed by geranylgeranyl group modification (prenylation) on theC-terminus of the Rac1 protein. Further, it has also been known that aRac1 protein interacts with RhoGDI via a geranylgeranyl group of theRac1 protein.

Hence, it was confirmed by a method described below that the method ofthe present invention enabled detection of a change in localization ofthese protein-protein interactions.

First, pRhoGDI-p62(PB1) was prepared by the same method as thatdescribed in Example 2 on the basis of a DNA sequence encoding the aminoacid sequence specified under Genbank ACCESSION No: NP_004300. Moreover,phAG-Rac1 and pp62(PB1)-PBD were as described in Example 21.

Then, each of the following combinations of the plasmid DNAs was mixedin equal amounts and introduced into cells by the same method as inExample 2:

a combination of phAG-Rac1 with pp62(PB1)-PBD; and

a combination of phAG-Rac1 with pRhoGDI-p62(PB1).

Then, after culturing for 6 hours after the transfection, an inhibitormevastatin (Enzo Life Sciences, Inc.) against geranylgeranyl groupmodification was added to the final concentration of 10 μM, allowed forfurther culturing for 15 hours, and observed. FIG. 40 shows the resultof the cells co-expressing AG-Rac1 and p62(PB1)-PBD. FIG. 41 shows theresult of the cells co-expressing AG-Rac1 and RhoGDI-p62(PB1). Note thatthe same cells are shown in each of FIGS. 40 and 41: (A) is photographedby the same method as that described in Example 1 using a normalinverted epifluorescence microscope; and (B) is photographed by the samemethod as that described in Example 17 using a total internal reflectionfluorescence microscopy system with arc lamp source.

As apparent from the result shown in the two upper panels of FIG. 40,the method of the present invention confirmed that, in a normal state,the protein-protein interaction between Rac1 and PBD took place bothinside and outside the nuclei; in other words, Rac1 was present in anactivated state.

It has been known that if geranylgeranyl group modification is inhibitedwith a drug such as mevastatin, Rac1 is localized in the nucleus.Regarding this knowledge, the method of the present invention alsoconfirmed as apparent from the result shown in the two lower panels ofFIG. 40 that the interaction between Rac1 and PBD was changed by themevastatin treatment, so that the interaction took place only in thenuclei. Further, by detecting the interaction, the method of the presentinvention also confirmed that Rac1 was present in an activated stateeven without the geranylgeranyl group modification.

Meanwhile, regarding the interaction between Rac1 and RhoGDI, the methodof the present invention confirmed as apparent from the result shown inthe two upper panels of FIG. 41 that, in a normal state, theprotein-protein interaction between Rac1 and RhoGDI took place outsidethe nuclei.

On the other hand, as apparent from the result shown in the two lowerpanels of FIG. 41, the method of the present invention confirmed thatRac1 was localized only in the nuclei by the mevastatin treatment asdescribed above, and further that since the geranylgeranyl groupmodification of Rac1 was suppressed, the interaction between Rac1 andRhoGDI was reduced.

Thus, the present invention confirmed that it was possible to detect howthe protein-protein interaction in multiple regions in the cell waschanged by a stimulus from the outside. Furthermore, it was confirmedthat it was also possible to detect the presence or absence of amodification of a protein influencing a protein-protein interaction (forexample, geranylgeranyl group modification of Rac1 in relation to aRac1-RhoGDI interaction).

Example 23

An interaction between KRas and cRaf is one of important signaltransductions for cell proliferation, differentiation, and so forth.Moreover, it has been revealed that this protein-protein interactiontakes place by an activation of KRas as a result of signaling viaGrb2-SOS from an EGF receptor activated by an epidermal growth factor(EGF). Further, it has also been known that this protein-proteininteraction changes the localization of cRaf from the cytoplasm to thecell membrane.

As described above, since the interaction between KRas and cRaf isdependent on EGF, the interaction does not take place in the absence ofEGF. However, among KRas mutants, there are also constitutively-activemutants (for example, KRasG12D) capable of interacting with cRaf in theabsence of EGF. Furthermore, such mutants have been detected in variouscancers. Thus, in the development of effective cancer treatments, and soforth, it is important to detect positional information and temporalinformation on a protein-protein interaction such as assembly formationbetween KRas or mutants thereof and cRaf, and localization of theassembly.

For this reason, whether or not the present invention enabled detectionof a difference in protein-protein interaction between a protein havinga disease-associated mutation and a wildtype protein thereof was testedby a method described below.

(Preparation of Plasmid DNAs)

pp62(PB1)-KRas(WT) was prepared by the same method as that described inExample 2 on the basis of a DNA sequence encoding the amino acidsequence specified under Genbank ACCESSION No: NP_004976.

Regarding pp62(PB1)-KRas(G12D), a mutation was introduced intopp62(PB1)-KRas(WT) by the same method as that described in Example 17using a primer having the DNA sequence of SEQ ID NO: 160(5′-CTTGTGGTAGTTGGAGCTGACGGCGTAGGCAAGAGTGCCTTG-3′). phAG-cRaf(R59A) wasprepared by the same method as that described in Example 2 on the basisof a DNA sequence (SEQ ID NO: 161) encoding a protein having the aminoacid sequence of SEQ ID NO: 162. Note that, as cRaf, a mutant of theprotein (cRaf(R59A)) was used in this Example with reference to themethod described in Harvey C D et al., Science, Jul. 4, 2008, vol. 321,no. 5885, pp. 136-140.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

The plasmid DNAs were mixed in equal amounts in the followingcombinations, and were introduced into Cos-7 cells by the same method asthat described in Example 2:

a combination of pp62(PB1)-KRas(WT) with phAG-cRaf(R59A); and

a combination of pp62(PB1)-KRas(G12D) with phAG-cRaf(R59A).

The cells were observed using a total internal reflection fluorescencemicroscopy system with arc lamp source (manufactured by OlympusCorporation, IX71-ARCEVA) capable of exciting only the vicinity of thecell membrane. Additionally, regarding the combination ofpp62(PB1)-KRas(WT) with the phAG-cRaf(R59A), an image was obtained whenno EGF was added. After that, EGF (manufactured by SIGMA CO.) was addedto the cells to the final concentration of 50 ng/ml and left standing at37° C. for 30 minutes. Then, the resultant was observed again. FIG. 42shows the obtained result.

As apparent from the result shown in FIG. 42, in the cells co-expressingp62(PB1)-KRas(WT) and AG-cRaf(R59A), no fluorescent focus (assemblyformation) was observed when no stimulus was applied from the outside(no EGF addition). Meanwhile, as EGF was added, the assembly formationwas observed at the cell membranes. On the other hand, in thecombination of pp62(PB1)-KRas(G12D) with phAG-cRaf(R59A), the assemblyformation was observed at the cell membranes even without a stimulusfrom the outside.

Thus, the present invention makes it possible to clearly understand adifference (such as dependency on external stimulus) of aprotein-protein interaction caused by a mutation associated with adisease. Therefore, the method of the present invention is effective inanalyzing the intracellular dynamics and the function of a protein andthe like associated with the disease.

Example 24 Detection 20 of Protein-Protein Interaction

As described in Example 19 also, it was confirmed by a method describedbelow that the present invention enabled detection of a change inprotein-protein interaction over time.

Note that, in Example 24, the targeted protein-protein interactionswere: an interaction between BclX(L) and Bak, and an interaction betweenBclX(L) and Bax. It has been revealed that both Bak and Bax interactwith BclX(L) via BH3 domains thereof. It has been revealed that thedissociation constant between BclX(L) and Bak BH3 domain is 340 nM, andthe dissociation constant between BclX(L) and Bax BH3 domain is 13 μM(see Sattler M et al., Science, Feb. 14, 1997, vol. 275, no. 5302, pp.983 to 986). Additionally, it has also been known that theseprotein-protein interactions are competitively inhibited by ABT-737 (BH3mimetic).

(Preparation of Plasmid DNAs)

pBak-hAG was prepared by the same method as that described in Example 2using a DNA (SEQ ID NO: 163) encoding a region having the amino acidsequence of SEQ ID NO: 164. phAG-Bax was prepared by the same method asthat described in Example 2 using a DNA (SEQ ID NO: 165) encoding aregion having the amino acid sequence of SEQ ID NO: 166. Moreover,pp62(PB1)-BclX(L) was as described in Example 20.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

The plasmid DNAs were mixed in equal amounts in the followingcombinations, and the genes were introduced into 293 cells by the samemethod as that described in Example 2:

a combination of pp62(PB1)-BclX(L) with pBak-hAG; and

a combination of pp62(PB1)-BclX(L) with phAG-Bax.

Then, to the 293 cells into these plasmid DNAs were introduced, ABT-737(manufactured by Santa Cruz Biotechnology, Inc.) was added to the finalconcentration of 15 μM, and images were obtained by the same method asthat described in Example 2 every 30 minutes for the cells co-expressingp62(PB1)-BclX(L) and Bak-AG, and every 5 minutes for the cellsco-expressing p62(PB1)-BclX(L) and AG-Bax. On the basis of the obtainedimages, analysis was performed by the same method as that described inExample 11, and a graph was prepared by plotting a total fluorescenceintensity of fluorescent foci (assemblies) against time. Note that, inthe graph, the x axis represents time (minutes), provided that time whenthe drug was added is 0, and the y axis represents the totalfluorescence intensity of fluorescent foci (assemblies). FIG. 43 showsthe result of the cells co-expressing p62(PB1)-BclX(L) and Bak-AG. FIG.44 shows the result of the cells co-expressing p62(PB1)-BclX(L) andAG-Bax.

As apparent from the results shown in FIGS. 43 and 44, in both of theinteraction between BclX(L) and Bak and the interaction between BclX(L)and Bax, the total fluorescence luminances of assemblies formed in amanner dependent on these protein-protein interactions were decreasedovertime by the addition of ABT-737. Thus, the present inventionverified that by detecting the fluorescent foci, it was possible todetect a period until the protein-protein interactions ended.

Moreover, the time when the total fluorescence luminance reached a halfvalue was approximately 80 minutes in the case of BclX(L) and Bak, andapproximately 35 minutes in the case of BclX(L) and Bax. Since the rateof decreasing assemblies was slower in BclX(L) and Bak with a smallerdissociation constant (dissociation constant: 340 nM) than in BclX(L)and Bax (dissociation constant: 13 μM), it was confirmed as describedabove that the present invention enabled evaluation of a strength of theprotein-protein interaction.

Example 25 Detection 21 of Protein-Protein Interaction

As described in Example 24 also, it was confirmed by a method describedbelow that the present invention enabled detection of a change over timein when a protein-protein interaction took place.

First, CHO-K1 cells stably expressing p62(PB1)-p53 and AG-MDM2 describedin Example 13 were seeded onto a 35-mm glass base dish (manufactured byAsahi Glass Co., Ltd.). Then, on the next day, Nutlin-3 (manufactured byCALBIOCHEM) was added thereto to the final concentration of 10 μM, andobserved by the same method as that described in Example 1. Images wereobtained 2 minutes before the addition and thereafter every 15 seconds.The obtained images were used to analyze a total fluorescence luminanceof assemblies by employing the same method as that described in Example11, and a graph was prepared by plotting the total fluorescenceluminance against time. FIG. 45 shows the obtained result.

As apparent from the result shown in FIG. 45, the total fluorescenceluminance of assemblies was decreased as soon as Nutlin-3 (manufacturedby CALBIOCHEM) was added. The time when the half value was reached wasapproximately 3 minutes. Thus, the present invention confirmed that, bydetecting the fluorescent foci, it was possible to detect a period untilthe protein-protein interaction ended and the process.

Example 26 Detection 22 of Protein-Protein Interaction

As described above, it was confirmed by a method described below thatthe present invention enabled detection of a change over time in when aprotein-protein interaction took place.

First, to HeLaS3 cells stably expressing mTOR(FRB domain)-AG andp62(PB1)-FKBP12 described in Example 11, rapamycin was added to thefinal concentration of 20 nM, and analyzed by the same method as thatdescribed in Example 11. FIG. 46 shows the obtained result.

As apparent from the result shown in FIG. 46, after rapamycin was added,assembly formation was induced as time elapsed. The time when the halfvalue was reached was approximately 3 minutes. Thus, it was confirmedthat the present invention enabled detection of time when theprotein-protein interaction took place and the process, by detecting thefluorescent foci.

Example 27 Detection 23 of Protein-Protein Interaction

It has been revealed that when ERK in cells is activated by an EGFstimulus, the ERK substrate (ERK_substrate) is phosphorylated; as aresult, the ERK_substrate and a ww domain of a Pint protein (Pin1(ww))interact with each other. Further, it has also been known that if a MEKinhibitor U0126 is added, the ERK activity is decreased; as a result,the ERK substrate is dephosphorylated, terminating the interactionbetween the ERK substrate and Pin1(ww).

In this Example, it was confirmed by a method described below that: thepresent invention enabled detection of an interaction between an ERKsubstrate and Pin1(ww) induced indirectly by EGF through ERK activation;the present invention enabled detection of an interaction between an ERKsubstrate and Pin1(ww) suppressed indirectly by U0126 through ERKinactivation; and the present invention enabled detection of an EGFstimulus-dependent signal transduction over time.

(Preparation of Plasmid DNA)

In this Example, in order to detect an EGF stimulus-dependentinteraction between an ERK substrate and Pin1(ww),pp62(PB1)-ERK_substrate-P2A-hAG-Pin1(ww)-NES was prepared with referenceto a system for detecting the interaction by utilizing FRET (seeChristopher D. Harvey et al., Proc Natl Acad Sci USA, Dec. 9, 2008, vol.105, nol. 49, pp. 19264 to 19269). Specifically,pp62(PB1)-ERK_substrate-P2A-hAG-Pin1(ww)-NES was prepared by the samemethod as that described in Example 2 using a chemically synthesized DNA(SEQ ID NO: 167) encoding a region having the amino acid sequence of SEQID NO: 168.

Note that, in the amino acid sequence of SEQ ID NO: 168, the amino acidsequence from positions 1 to 102 shows the amino acid sequence ofp62(PB1). The amino acid sequence from positions 103 to 128 shows alinker sequence. The amino acid sequence from positions 129 to 138 showsthe amino acid sequence from positions 43 to 52 of human Cdc25C that isthe ERK substrate, and the amino acid sequence from positions 139 to 142shows the amino acid sequence of an ERK docking site. The amino acidsequence from positions 146 to 164 shows the amino acid sequence of aP2A peptide. The amino acid sequence from positions 165 to 390 shows theamino acid sequence of AG. The amino acid sequence from positions 391 to416 shows a linker sequence. The amino acid sequence from positions 417to 470 shows the amino acid sequence of Pin1(ww). The amino acidsequence from positions 471 to 482 shows the amino acid sequence of anuclear export signal (NES) of MEK.

Moreover, the P2A peptide inserted between p62(PB1)-ERK_substrate andAG-Pin1(ww)-NES is a CHYSEL (cis-acting hydrolase element) sequencederived from porcine teschovirus. It has been known that when theprotein is translated, ribosome skipping occurs, generating cleavage infront of proline at the end of the amino acid sequence(ATNFSLLKQAGDVEENPGP) (see Donnelly M L et al., J Gen Virol., may 2001,vol. 82, no. 5, pp. 1013 to 1025). Thus, if thepp62(PB1)-ERK_substrate-P2A-hAG-Pin1(ww)-NES is introduced into cells,this is consequently cleaved into: a product having a portion of the P2Apeptide fused to the C-terminus of p62(PB1)-ERK_substrate; and a producthaving a portion of the P2A peptide fused to the N-terminus ofAG-Pin1(ww)-NES. The two are expressed in the cells.

(Transfection into Cultured Cells, and Observation of Transfected Cells)

pp62(PB1)-ERK_substrate-P2A-hAG-Pin1(ww)-NES was transfected into 293cells by the same method as that described in Example 1. On the nextday, EGF (manufactured by SIGMA CO.) was added to the cells to the finalconcentration of 50 ng/ml. Further, 14 minutes thereafter, U0126 wasadded to the cells to the final concentration of 10 μM. Meanwhile, thecell observation was started 2 minutes before the EGF addition, and theobserved images were obtained every 15 seconds. The obtained images wereused to analyze a total fluorescence luminance of fluorescent foci(assemblies) by the same method as that described in Example 11, and agraph was prepared by plotting the total fluorescence luminance againsttime. FIG. 47 shows the obtained result.

As apparent from the result shown in FIG. 47, in the cells expressingp62(PB1)-ERK_substrate and AG-Pin1(ww)-NES, after EGF was added,fluorescent foci (assemblies) were significantly observed as timeelapsed. However, after U0126 was added, the fluorescent foci wereslowly decreased, reflecting the promotion of ERK substratedephosphorylation in response to endogenous ERK inactivation due to theaddition.

The change over time in the total fluorescence luminance of fluorescentfoci (assemblies) representing the interaction between the ERK substrateand Pin1(ww) was substantially the same as the result obtained bymeasuring an ERK substrate-Pin1(ww) interaction using FRET described inChristopher D. Harvey et al., Proc Natl Acad Sci USA, Dec. 9, 2008, vol.105, no. 49, pp. 19264 to 19269.

In this manner, the present invention makes it possible to detect aprotein-protein interaction induced or inhibited indirectly by aparticular stimulus, by detecting fluorescent foci. Moreover, thepresent invention also makes it possible to detect a signal transductionover time.

Example 28 Detection 24 of Protein-Protein Interaction

As described above, based on the interaction between HRas and cRafdescribed in Example 20, it was confirmed that the present inventionenabled detection of a change over time in when a protein-proteininteraction took place.

Specifically, pp62(PB1)-HRas(WT) described in Example 20 andphAG-cRaf(R59A) described in Example 23 were introduced into cells bythe same method as that described in Example 23. EGF (manufactured bySIGMA CO.) was added thereto to the final concentration of 50 ng/ml. Ameasurement apparatus capable of detecting a fluorescent signal only inthe vicinity of the cell membrane was used for the observation. Notethat the observation was started 5 minutes before the EGF addition, andcontinued at 5-minute intervals for 30 minutes after the addition. Theobservation was made another 30 minutes thereafter. On the basis of theobtained image data, analysis was performed by the same method as thatdescribed in Example 11, and a graph was prepared by plotting a totalfluorescence intensity of fluorescent foci (assemblies) against time.Note that, in the graph, the x axis represents time (minutes), providedthat time when EGF was added is 0, and the y axis represents the totalfluorescence intensity of fluorescent foci (assemblies). FIG. 48 showsthe obtained result.

As apparent from the result shown in FIG. 48, the interaction betweenHRas and cRaf that took place by adding EGF was successfully detectedover time.

Moreover, as described above, it has been known that cRaf interacts withHRas activated as a result of signaling via a Grb2-SOS complex from anEGF receptor of cells to which EGF has been added. Thus, the resultshown in FIG. 48 confirmed that the present invention enabled tracingover time of a process in which an intracellular signal transductionpathway (such as a signal transduction pathway via a Grb2-SOS complex)was activated in response to a stimulus (such as EGF) applied to cellsfrom the outside.

Example 29 Detection 25 of Protein-Protein Interaction

As described above, it has been revealed that rapamycin binds to aFKBP12 protein, and this complex further binds to a FRB domain of a mTORprotein (mTOR(FRB)). A mTOR protein is a serine/threonine kinase havinga function of activating signal transductions involved in proteinsynthesis and cell proliferation. It has also been revealed that thefunction is inhibited by such complex formation between a FKBP12 proteinand rapamycin.

Furthermore, a FKBP12 protein has been known to interact with a proteinphosphatase (calcineurin) composed of a catalytic subunit A (calcineurinA) and a regulatory subunit B (calcineurin B) via FK506. Calcineurin isan enzyme having a very important function in signal transductions in Tcells and the like. It has also been revealed that the function isinhibited by such complex formation between a FKBP12 protein and FK506.

Hence, in this Example, a test was conducted by a method described belowregarding whether or not the present invention enabled detection anddistinguishment of: a complex of FKBP12 and mTOR(FRB) formed in a mannerdependent on rapamycin in a single cell in which FKBP12, mTOR(FRB),calcineurin A and calcineurin B were co-expressed; a complex of FKBP12with calcineurin A and calcineurin B formed in a manner dependent onFK506; and eventually an inhibition of signal transductions in whichthese complexes were involved.

Note that as the “calcineurin A and calcineurin B” expressed in thecell, a mCAB protein was used, which was composed of a portion ofcalcineurin A fused to a portion of calcineurin B (see Clemons P A etal., Chem Biol., January 2002, vol. 9, iss. 1, pp. 49 to 61).

First, phAG-mCAB, pp62(PB1)-FKBP12, and pmTOR(FRB)-hKO1 were introducedinto HeLaS3 cells. The phAG-mCAB was prepared by the same method as thatdescribed in Example 2 using an artificially synthesized DNA (SEQ ID NO:169) encoding a region having the amino acid sequence of SEQ ID NO; 170.The pp62(PB1)-FKBP12 was as described in Example 2. The pmTOR(FRB)-hKO1(pmTOR(FRB domain)-hKO1) was as described in Example 6.

Moreover, the HeLaS3 cells were cultured by the same method as thatdescribed in Example 1. Further, in the transfection, the HeLaS3 cellswere seeded onto 2 wells of an 8-well chamber (manufactured by NuncA/S). On the next day, by the same method as that described in Example1, 130 ng of each of the plasmid DNAs was introduced into the HeLaS3cells using 1 μl of Transfection Reagent.

Then, 24 hours thereafter, the transfected cells were observed by thesame method as that described in Example 1. To the cells, rapamycin orFK506 was added to the final concentration of 500 nM, and observedanother 15 minutes thereafter. FIG. 49 shows the obtained result.

As apparent from the result shown in FIG. 49, by adding rapamycin to thecells expressing AG-mCAB, p62(PB1)-FKBP12, and mTOR(FRB)-KO1, theinteraction between mTOR(FRB) and FKBP12 was observed in the form offluorescent foci emitting a KO1-derived fluorescent signal. On the otherhand, by adding FK506, the interaction between mCAB and FKBP12 wasobserved in the form of fluorescent foci emitting an AG-derivedfluorescent signal.

Thus, it was confirmed that the present invention enabled detection ofmultiple types of protein-protein interactions in a single cell,particularly, various protein-protein interactions dependent ondifferent stimuli in a single cell.

Moreover, the present invention can provide a method for detecting anddistinguishing multiple types of signal transductions in a single cellby detecting various protein-protein interactions involved in signaltransductions in a single cell.

Furthermore, as described in this Example also, unlike FRET that isanother method for detecting a protein-protein interaction in livingcells, it is not necessary to select a fluorescent protein meeting theconditions of an acceptor and a donor; in addition, it is not necessaryto take into consideration cross excitation by which an acceptorfluorescent protein is excited, and bleed-through in which fluorescenceof a donor fluorescent protein bleeds through a filter (absorptionfilter) set for detecting fluorescence of an acceptor fluorescentprotein. Thus, in the present invention, combinations of variousfluorescent proteins having different wavelength characteristics can beeasily selected and utilized.

Example 30 Detection 26 of Protein-Protein Interaction

Whether or not the method of the present invention enabled detection ofknown protein-protein interactions shown in Table 3 by using p62(PB1) asthe association-inducing protein and an AG protein as the fluorescentprotein having a multimerization ability was tested by the methoddescribed in Example 2.

TABLE 3 Protein-protein interaction Cdk5 P25 Plk Wee1 calcineurin AαVIVIT peptide JNK JIP CREB CBP ERK2 MEK MEK cRaf β-catenin TCF

As a result, although unillustrated, it was verified that it waspossible to detect the protein-protein interactions in all thecombinations in the form of fluorescent foci. It was demonstrated thatthe present invention was a generally-adoptable method for detecting aprotein-protein interaction.

INDUSTRIAL APPLICABILITY

As has been described above, the present invention makes it possible todetect a protein-protein interaction in an intracellular environmentunique thereto, and to detect positional information and temporalinformation on the protein-protein interaction. Moreover, in the presentinvention, a strength of a protein-protein interaction correlates withthe fluorescence intensity of a fluorescent focus. Accordingly, themethod is utilizable in identifying an amino acid residue involved in aprotein-protein interaction, and also in screening for a substancemodulating a protein-protein interaction, on the basis of thefluorescence intensity.

Thus, the method for detecting a protein-protein interaction and soforth of the present invention and a kit for use in these methods areuseful in the development of pharmaceutical products and so on throughelucidations of various signal transductions in vivo, various biologicalreaction controls, and the like, and eventually through elucidations ofdisease mechanisms.

[Sequence Listing Free Text]

SEQ ID NOs: 1 and 2

<223> humanized-codon Azami Green (AG)

SEQ ID NOs: 3 and 4

<223> PB1 domain of p62

SEQ ID NOs: 5 and 6

<223> PB1 domain of MEK5

SEQ ID NOs: 7 and 8

<223> PB1 domain of Nbr1

SEQ ID NOs: 9 and 10

<223> PB1 domain of PKCiota

SEQ ID NOs: 11 and 12

<223> PB1 domain of TFG

SEQ ID NOs: 13 and 14

<223> SAM domain of TEL

SEQ ID NOs: 15 and 16

<223> SAM domain of EphB2

SEQ ID NOs: 17 and 18

<223> SAM domain of DGK delta

SEQ ID NOs: 19 and 20

<223> SAM domain of Tankyrase-1

SEQ ID NOs: 21 and 22

<223> FRB domain of mTOR

SEQ ID NOs: 23 and 24

<223> FKBP12

SEQ ID NOs: 25 and 26

<223> p53

SEQ ID NOs: 27 and 28

<223> MDM2

SEQ ID NOs: 29 and 30

<223> Sec5

SEQ ID NOs: 31 and 32

<223> RalB

SEQ ID NOs: 33 and 34

<223> RalB protein Q72L mutant

SEQ ID NOs: 35 and 36

<223> RalB protein S28N mutant

SEQ ID NOs: 37 and 38

<223> calmodulin

SEQ ID NOs: 39 and 40

<223> M13 peptide

SEQ ID NOs: 41 and 42

<223> HRas

SEQ ID NOs: 43 and 44

<223> cRaf

SEQ ID NOs: 45 and 46

<223> Smac

SEQ ID NOs: 47 and 48

<223> XIAP

SEQ ID NOs: 49 and 50

<223> BclX(L)

SEQ ID NOs: 51 and 52

<223> BAD

SEQ ID NOs: 53 and 54

<223> Rac1

SEQ ID NOs: 55 and 56

<223> PBD

SEQ ID NO: 57

<223> artificially synthesized hAG forward primer 1 sequence

SEQ ID NO: 58

<223> artificially synthesized hAG reverse primer 1 sequence

SEQ ID NO: 59

<223> artificially synthesized p62(PB1) forward primer 1 sequence

SEQ ID NO: 60

<223> artificially synthesized p62(PB1) reverse primer 1 sequence

SEQ ID NO: 61

<223> artificially synthesized hAG forward primer 2 sequence

SEQ ID NO: 62

<223> artificially synthesized hAG reverse primer 2 sequence

SEQ ID NO: 63

<223> artificially synthesized p62(PB1) forward primer 2 sequence

SEQ ID NO: 64

<223> artificially synthesized p62(PB1) reverse primer 2 sequence

SEQ ID NO: 65

<223> artificially synthesized p62(PB1) forward primer 3 sequence

SEQ ID NO: 66

<223> artificially synthesized p62(PB1) reverse primer 3 sequence

SEQ ID NO: 67

<223> artificially synthesized mTOR(FRB) forward primer sequence

SEQ ID NO: 68

<223> artificially synthesized mTOR(FRB) reverse primer sequence

SEQ ID NO: 69

<223> artificially synthesized FKBP12 forward primer sequence

SEQ ID NO: 70

<223> artificially synthesized FKBP12 reverse primer sequence

SEQ ID NO: 71

<223> artificially synthesized MEK(PB1) forward primer sequence

SEQ ID NO: 72

<223> artificially synthesized MEK(PB1) reverse primer sequence

SEQ ID NO: 73

<223> artificially synthesized Nbr1(PB1) forward primer sequence

SEQ ID NO: 74

<223> artificially synthesized Nbr1(PB1) reverse primer sequence

SEQ ID NO: 75

<223> artificially synthesized PKCiota(PB1) forward primer sequence

SEQ ID NO: 76

<223> artificially synthesized PKCiota(PB1) reverse primer sequence

SEQ ID NO: 77

<223> artificially synthesized TFG(PB1) forward primer sequence

SEQ ID NO: 78

<223> artificially synthesized TFG(PB1) reverse primer sequence

SEQ ID NO: 79

<223> artificially synthesized TEL(SAM) forward primer sequence

SEQ ID NO: 80

<223> artificially synthesized TEL(SAM) reverse primer sequence

SEQ ID NO: 81

<223> artificially synthesized EphB2 (SAM) forward primer sequence

SEQ ID NO: 82

<223> artificially synthesized EphB2(SAM) reverse primer sequence

SEQ ID NO: 83

<223> artificially synthesized DGK delta(SAM) forward primer sequence

SEQ ID NO: 84

<223> artificially synthesized DGK delta(SAM) reverse primer sequence

SEQ ID NO: 85

<223> artificially synthesized Tankyrase(SAM) forward primer sequence

SEQ ID NO: 86

<223> artificially synthesized Tankyrase(SAM) reverse primer sequence

SEQ ID NO: 87

<223> artificially synthesized TFG(PB1) forward primer 2 sequence

SEQ ID NO: 88

<223> artificially synthesized TFG(PB1) reverse primer 2 sequence

SEQ ID NO: 89

<223> artificially synthesized TEL(SAM) forward primer 2 sequence

SEQ ID NO: 90

<223> artificially synthesized TEL(SAM) reverse primer 2 sequence

SEQ ID NO: 91

<223> artificially synthesized DGK delta(SAM) forward primer 2 sequence

SEQ ID NO: 92

<223> artificially synthesized DGK delta(SAM) reverse primer 2 sequence

SEQ ID NO: 93

<223> artificially synthesized Tankyrase(SAM) forward primer 2 sequence

SEQ ID NO: 94

<223> artificially synthesized Tankyrase(SAM) reverse primer 2 sequence

SEQ ID NO: 95

<223> artificially synthesized hKO1 forward primer sequence

SEQ ID NO: 96

<223> artificially synthesized hKO1 reverse primer sequence

SEQ ID NO: 97

<223> artificially synthesized p53 forward primer sequence

SEQ ID NO: 98

<223> artificially synthesized p53 reverse primer sequence

SEQ ID NO: 99

<223> artificially synthesized MDM2 forward primer sequence

SEQ ID NO: 100

<223> artificially synthesized MDM2 reverse primer sequence

SEQ ID NO: 101

<223> artificially synthesized Sec5 forward primer sequence

SEQ ID NO: 102

<223> artificially synthesized Sec5 reverse primer sequence

SEQ ID NO: 103

<223> artificially synthesized RalB forward primer sequence

SEQ ID NO: 104

<223> artificially synthesized RalB reverse primer sequence

SEQ ID NO: 105

<223> artificially synthesized RalB(Q72L) mutation primer sequence

SEQ ID NO: 106

<223> artificially synthesized RalB(S28N) mutation primer sequence

SEQ ID NO: 107

<223> artificially synthesized calmodulin forward primer sequence

SEQ ID NO: 108

<223> artificially synthesized calmodulin reverse primer sequence

SEQ ID NO: 109

<223> artificially synthesized M13 peptide forward primer sequence

SEQ ID NO: 110

<223> artificially synthesized M13 peptide reverse primer sequence

SEQ ID NO: 111

<223> artificially synthesized AGNLS forward primer sequence

SEQ ID NO: 112

<223> artificially synthesized AGNLS reverse primer sequence

SEQ ID NO: 113

<223> artificially synthesized HRas forward primer sequence

SEQ ID NO: 114

<223> artificially synthesized HRas reverse primer sequence

SEQ ID NO: 115

<223> artificially synthesized KRas forward primer sequence

SEQ ID NO: 116

<223> artificially synthesized KRas reverse primer sequence

SEQ ID NO: 117

<223> artificially synthesized HRas mutant primer sequence

SEQ ID NO: 118

<223> artificially synthesized cRaf forward primer sequence

SEQ ID NO: 119

<223> artificially synthesized cRaf reverse primer sequence

SEQ ID NO: 120

<223> artificially synthesized Smac forward primer sequence

SEQ ID NO: 121

<223> artificially synthesized XIAP forward primer sequence

SEQ ID NO: 122

<223> artificially synthesized XIAP reverse primer sequence

SEQ ID NO: 123

<223> artificially synthesized BclX(L) forward primer sequence

SEQ ID NO: 124

<223> artificially synthesized BclX(L) reverse primer sequence

SEQ ID NO: 125

<223> artificially synthesized BAD forward primer 1 sequence

SEQ ID NO: 126

<223> artificially synthesized BAD forward primer 2 sequence

SEQ ID NO: 127

<223> artificially synthesized BAD reverse primer sequence

SEQ ID NO: 128

<223> artificially synthesized Rac1 forward primer sequence

SEQ ID NO: 129

<223> artificially synthesized Rac1 reverse primer sequence

SEQ ID NO: 130

<223> artificially synthesized PBD forward primer sequence

SEQ ID NO: 131

<223> artificially synthesized PBD reverse primer sequence

SEQ ID NO: 132

<223> base sequence of monomeric KO (Kusabira-Orange)

SEQ ID NO: 133

<223> amino acid sequence of monomeric KO (Kusabira-Orange)

SEQ ID NOs: 134 and 135

<223> humanized-codon mAG1

SEQ ID NOs: 136 and 137

<223> mMiCy1

SEQ ID NOs: 138 and 139

<223> mKikGR1

SEQ ID NOs: 140 and 141

<223> KCy1

SEQ ID NOs: 142 and 143

<223> dAG (AB)

SEQ ID NOs: 144 and 145

<223> dAG (AC)

SEQ ID NOs: 146 and 147

<223> TGuv

SEQ ID NOs: 148 and 149

<223> Momiji

SEQ ID NOs: 150 and 151

<223> COR3.01

SEQ ID NOs: 152 and 153

<223> DsRed2

SEQ ID NOs: 154, 159, and 160

<223> artificially synthesized primer sequences

SEQ ID NOs: 155, 157, 161, 163, 165, 167, and 169

<223> artificially synthesized polynucleotide sequences

SEQ ID NOs: 156, 158, 162, 164, 166, 168, and 170

<223> artificially synthesized polypeptide sequences

SEQ ID NOs: 171 and 172

<223> COR5

The invention claimed is:
 1. A method for detecting an interactionbetween a first protein and a second protein, the method comprising thesteps of: expressing in a cell a first fusion protein comprising thefirst protein and an association-inducing protein, and a second fusionprotein comprising the second protein and a fluorescent protein having amultimerization ability and able of emitting fluorescence; determiningthat there exists an interaction between the first protein and thesecond protein by detecting a fluorescent focus, wherein theassociation-inducing protein is at least one protein selected from thegroup consisting of PB1 domain of p62, a PB1 domain of TFG, a PB1 domainof PKCiota, a SAM domain of TEL, a SAM domain of DGK delta, and a SAMdomain of Tankyrase-1; the fluorescent protein having a multimerizationability is at least one fluorescent protein selected from the groupconsisting of a fluorescent protein capable of forming a homomultimer,monomeric Kusabira-Orange 2, monomeric Keima-Red, monomericMidoriishi-Cyan1, monomeric Kusabira-Orange 1, and monomeric KikumeGreen-Red1; and the fluorescent focus has a fluorescence intensity in aregion of 0.2 to 5 μm, the fluorescence intensity being higher than afluorescence intensity of the fluorescent protein which is present in adispersed state in the cell, and wherein the association inducingprotein is able to form a fluorescent focus when fused to thefluorescent protein having an multimerization ability.
 2. The method ofclaim 1, further detecting the presence of the interaction between thefirst protein and the second protein for a period of time wherein theabsence of the focus indicates the ending of the interaction.
 3. Themethod of claim 2, further comprising adding a stimulus to the cell. 4.The method of claim 3, wherein the interaction ends in response to astimulus and further comprising measuring a time period from applyingthe stimulus to the cell to extincting the focus.